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Key value for chemical safety assessment

Effects on fertility

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Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Jan 2009 - 21 Aug 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance glycerides, castor-oil, mono, hydrogenated, acetates (CAS 736150-63-3). According to the ECHA guidance document ‘Practical guide 6: How to report read-across and categories' (ECHA, 2012), the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 426 (Developmental Neurotoxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF, Testing Guidelines for Toxicity Studies (2-1-17), 12 Nohsan No 8147, 2000-11-24
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, UK
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley Crl:CD® (SD) IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, UK
- Age at study initiation: (P) 7-8 weeks
- Weight at study initiation: (P) 216-361 g (males) and 149-249 g (females)
- Housing: All P animals were housed in groups of up to 4 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper. Mated females were housed individually (or with their litter), in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes (Datesand Ltd., Cheshire, UK). For the offspring selected to form the F1 generation, this housing procedure was repeated. The P and F1 offspring selected for assessment of developmental neurotoxicity were housed in groups of up to 4 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding.
- Diet: ground diet (Rodent PMI 5002 Diet, BCM IPS Limited, London, UK), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Each concentration of the test material was prepared by weighing an appropriate amount and mixing it with a small amount of the required volume of diet. Once this was adequately mixed the formulation was then transferred to a Hobart H800 mixer and mixed with the remaining required volume of diet.

DIET PREPARATION
- Mixing appropriate amounts with: laboratory diet

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating.
- After 14 days of unsuccessful pairing replacement of first male by another male or extension of the mating phase to a 3 week.
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test material in the dietary admixtures was determined by gas chromatography using an external standard technique. The dietary admixtures were sampled and analysed within 2 weeks of preparation. Homogeneity and stability determinations were performed under Harlan Laboratories Ltd. project number 2384/0006. The results showed that achieved concentrations were between 95 and 121% of nominal concentration.
Duration of treatment / exposure:
(P) Males: 10 weeks during maturation and throughout mating, gestation and until completion of the P female lactation phase.
(P) Females: 10 weeks during maturation and throughout mating, gestation and until Day 21 lactation phases.
(F1) Females: minimum of 10 weeks during maturation and subsequently throughout mating, gestation and lactation phases. Between weaning and the formal start of the F1 generation, the animals continued to receive their appropriate treated diet.
(F1) Males: minimum of 10 weeks during maturation and subsequently throughout mating, gestation and until completion of the F1 female lactation phases. Between weaning and the formal start of the F1 generation, the animals continued to receive their appropriate treated diet.
(F2) Males/ Females: from weaning (Day 21 of age) to termination at Day 70 of age.
Frequency of treatment:
daily, 7 days/week
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 10 weeks
- Due to number of offspring required for developmental neurotoxicity, the study was split into two equal parts. Each group was divided into two equal sub-groups; with animals allocated to the second part being given treated diets eight days later. Chronically, all procedures occurred approximately one week later for the second sub-group compared to the first group.
Remarks:
Doses / Concentrations:
1500, 6000 and 25000 ppm
Basis:
other: nominal in diet: animals in the high dose group initially received 15000 ppm rising to 20000 ppm to 25000 ppm during the maturation phase of each generation
Remarks:
Doses / Concentrations:
82, 324 and 1159 mg/kg bw/day
Basis:
other: mean achieved dose level (P males)
Remarks:
Doses / Concentrations:
146, 587 and 2200 mg/kg bw/day
Basis:
other: mean achieved dose level (P females)
Remarks:
Doses / Concentrations:
109, 435 and 1342 mg/kg bw/day
Basis:
other: mean achieved dose level (F1 males)
Remarks:
Doses / Concentrations:
160, 630 and 2262 mg/kg bw/day
Basis:
other: mean achieved dose level (F1 females)
No. of animals per sex per dose:
28 P males, 28 P females
24 F1 males, 24 F1 females
Control animals:
other: yes, laboratory diet treated with Arachis oil to ensure comparable calorific intake
Details on study design:
- Dose selection rationale: The dietary levels were based on known toxicology data including an earlier preliminary study in the rat (Project number 2384/0006).
- Other: In each generation, animals receiving the high dose level initially received the test material at a concentration of 15000 ppm and this was increased to 20000 ppm and finally 25000 ppm as the study progressed. Animals were receiving the highest inclusion level by the end of the maturation/pre-pairing phase in both generations and therefore were receiving the higher dietary inclusion by the time of the main reproductive assessment.
Positive control:
The study incorporated a positive control for endocrine disruption, DEHP (bis(2-thylhexyl) phthalate), throughout the P generation and until sexual maturation of the F1 ofspring. Animals received an intended dietary inclusion level of 10000 ppm.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the week and once daily on weekends and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1, prior to treatment, weekly throughout the maturation phase of each generation and continuing for males until termination. Following pairing P/F1 females were weighted daily until mating was evident. Mated females were weighted on Day 0, 7, 14 and 21 post coitum and for females that littered on Day 1, 4, 7, 14 and 21 post partum. P/F1 offspring (selected for post-weaning assessment of developmental neurotoxicity) were weighted on Day 28, 35, 42, 49, 56, 63 and 70 of age. Body weight was recorded for all animals on the Day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption was assessed for each cage of adults during the maturation period and for males after mating/pairing. Females showing evidence of mating, food consumption was recorded for the periods Day 0-7, 7-14 and 14-21 post coitum. For females that littered, food consumption was recorded for the period covering Day 1-4, 4-7, 7-14 and 14-21 post partum.

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: daily by visual inspection for any overt change
Oestrous cyclicity (parental animals):
Prior to pairing of females for the P and F1 mating phases, a vaginal smear was taken daily for twenty-one days and a sample was placed on a glass slide. The smears were allowed to dry and then stained using a diluted giemsa stain and examined microscopically.
Sperm parameters (parental animals):
Parameters examined in P/F1 male parental generations in control and high-dose males: Yes. Testis weight, epididymis weight, spermatid enumeration, sperm motility, sperm morphology.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in P/ F1 offspring: number and sex of pups, live births, postnatal mortality reported on Day 1, 4, 7, 14 and 21, clinical condition, individual bodyweight, necropsy findings, ano-genital distance and number of visible nipples on Day 11 and 15 and neurobehavioural effects.

GROSS EXAMINATION OF DEAD PUPS:
Yes, dead offspring was subjected to a necropsy examination.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving male F0/F1 animals were killed following completion of the F0 female lactation phase.
- Maternal animals: All surviving F0/F1 females were killed and examined macroscopically at Day 21 of lactation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Selected organs (adrenals, coagulating gland, right epididymis, ovaries, right testis, pituitary, prostate, seminal vesicles, uterus with oviducts and cervix, vagina, gross lesions) were weighed and/or preserved for histopathological examination.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- The P and F1 offspring selected for the post-weaning-developmental neurotoxicity were killed at Day 70 of age.
- All unselected offspring and those dying during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS
The following tissues were preserved from all F1 males and females from each dose group, in buffered 10% formalin: adrenals, coagulating gland, right epididymis, ovaries, right testis, pituitary, prostate, seminal vesicles, uterus with oviducts and cervix, vagina, gross lesions. The tissues from all control and high-dose animals and any treated animals which failed to mate or that did not achieve a pregnancy were processed. In addition, the following tissues were prepared for microscopic examination and weighed, respectively. The brain, spleen, thymus and uterus were weighed and preserved for one unselected male/female F1/F2 offspring at necropsy. In addition, samples of the following tissues of perfused animals at Day 70 of age (selected for the post-weaning-developmental neurotoxicity examinations) were histopathologically investigated: brain, dorsal root ganglia, dorsal and ventral root fibres, eyes, optic nerve, peroneal nerve, sciatic nerve, sural nerve, tibial nerve, skeletal muscle and spinal cord.
Statistics:
Data were initially assessed for homogeneity of variance using Levene´s test. Where variances were shown to be homogenous, a parametric assessment of the data was performed using one way analysis of variance (ANOVA), which if significant was followed by pairwise comparisons using Dunnett´s test. Where Levene´s test showed unequal variances, the data were analysed using non-parametric methodology: Kruskal-Wallis ANOVA which, if significant, was followed by Mann-Whitney U-test. Probability values (p) are presented as follows: p ≤ 0.001***, p ≤ 0.01**, p ≤ 0.05*, p > 0.05 (not significant).
Reproductive indices:
Mating performance and fertility
- Pre-coital interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

Fertility Indices
- Mating Index (%): (Number of animals mated/ number of animals paired) x 100
- Pregnancy Index (%): (Number of pregnant females/ number of females mated) x 100

Gestation and Parturition Data
- Gestation length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition. Where the start of parturition occurred overnight, the total was adjusted by adding half a day.
- Parturition Index (%): (Number of females delivering live offspring/ number of pregnant females) x 100

- Sex Ratio (% males): (Number of male offspring/ total number of offspring) x 100
Offspring viability indices:
Lactation Data
- Implantation losses (%):
% pre-implantation loss = [(Number of corpora lutea - number of implantation sites) / number of corpora lutea] x 100
% post-implantation loss = [(Group number of implantation sites - number of offspring) / number of implantation sites] x 100
offspring) x 100

Live Birth and Viability Indices
- Live Birth Index (%) = (Number of offspring alive on Day 1/ number of offspring born) x 100
- Viability Index 1 (%) = (Number of offspring alive on Day 4/ number of offspring alive on Day 1) x 100
- Viability Index 2 (%) = (Number of offspring alive on Day 7/ number of offspring alive on Day 4) x 100
- Viability Index 3 (%) = (Number of offspring alive on Day 14/ number of offspring alive on Day 7) x 100
- Viability Index 4 (%) = (Number of offspring alive on Day 21/ number of offspring alive on Day 14) x 100
- Viability Index 5 (%) = (Number of offspring alive on Day 21/ number of offspring alive on Day 1) x 100

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
25000 ppm: one P female was killed due to a decline in clinical condition; control/6000 ppm: one P female in each group was killed due to adverse clinical condition around the time of expected parturition, not treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1500/25000 ppm: increase in bodyweight gain during Days 1-21; 25000 ppm (P, female): significant lower food intake during the last week of lactation; 6000 ppm (P, female): higher food intake during last week of gestation and lactation, non-adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1500/25000 ppm: increase in bodyweight gain during Days 1-21; 25000 ppm (P, female): significant lower food intake during the last week of lactation; 6000 ppm (P, female): higher food intake during last week of gestation and lactation, non-adverse.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
6000 ppm/control: one P female animal of each group showed an irregular cycle, non-adverse.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
25000 ppm: lower homogenisation resistant spermatid counts (P amles) for the epididymides and testes, non-adverse.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
25000 ppm: lower number of P females with live offspring, higher post-implantation loss, not treatment-related.
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no treatment-related deaths amongst parental animals. One female in the high-dose group showed dehydration, lethargy, emaciation, hunched posture, tip-toed gait and staining around mouth. Macroscopic necropsy examination indicated that the animal had a suspected broken jaw and the decline in clinical condition was considered to be unrelated to treatment. One control P female showed hunched posture, pilo-erection, tip-toed gait, lethargy pallor of the extremities and vaginal discharge on Day 97 of the P generation. One female in the mid-dose group showed hunched posture, pilo-erection, staining around the snout, staining around ano-genital region and pallor of the extremities on Day 99 of the P generation. These clinical signs coincided with the time of expected parturition and were considered to reflect difficulties with the birth of their litter. These animals were therefore killed for animal welfare considerations.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No adverse effects of treatment on mean bodyweight or bodyweight change was apparent in either generation for males and females. A significant increase in bodyweight gain during Days 1-21 in the low- and high-dose female P group was considered to be not treatment related.
No adverse effect of treatment on mean food consumption was apparent in males and females. Food consumption of P females in the high-dose group was significantly lower during the last week of lactation. In mid-dose P females a higher food intake was observed during the last week of gestation and lactation.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Achieved intakes for animals were in line with expectations throughout the P and F1 generations (see Table 1 under “Any other information on results incl. tables”).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no obvious adverse effects of treatment on the oestrous cycles of females. One control and one mid-dose animal of the P generation showed an irregular cycle. There was no adverse effect of treatment on corpora lutea count, numbers of implantation sites or litter size at birth for females receiving the test substance. The mean number of small, medium or large oocyte follicles in the ovaries for F1 females of the high-dose group did not indicate any adverse effects of treatment (see Table 2 under “Any other information on results incl. tables”).

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No adverse effects of treatment on sperm concentration, motility or morphology were apparent for males receiving the test substance in either generation. For P males at the high-dose, homogenisation resistant spermatid counts for the epididymides and testes were lower than concurrent control, this decrease was considered to be incidental and of no toxicological significance (see Table 3 under “Any other information on results incl. tables”).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no obvious adverse effects of treatment on mating performance, fertility or gestation length of females receiving the test substance. While the number of P females with live offspring was marginally lower than anticipated in the high-dose group, there was no similar occurrence in the following F1 generation and this finding was therefore considered coincidental and unrelated to treatment.
In total there were 26, 25, 26 and 23 P females at 0 (control), 1500, 6000 and 25000 ppm of the test substance, respectively, that successfully reared young to weaning. In P females at the high-dose group a higher post-implantation loss was observed (see Table 3 under “Any other information on results incl. tables”). There was no subsequent significant difference in litter size at birth for the P females at the high-dose and no similar increase in post-implantation loss for F1 females at this dosage. In view of this, the higher implantation loss observed was considered to be incidental and unrelated to treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
For P males receiving the mid-dose, absolute and bodyweight-relative organ weight for the left testis was significantly higher than control. In the absence of any significant increase for the right testis for these males or both testes for F1 males or any treatment related histopathological change, this effect was considered to be incidental and unrelated to treatment. For P females of the high-dose group, absolute and bodyweight-relative thyroid weights were significantly higher. There were no significant differences in thyroid weights for F1 females or for males in either generation at this inclusion level. Given the earlier and potentially higher exposure of the F1 females to the test substance (due to low bodyweight at this stage) and the absence of any accompanying effect on thyroid weights for these animals, this finding was therefore considered to be incidental and of no toxicological significance. For P females receiving the mid and high-dose, absolute uterine weight was significantly lower; values when adjusted for the bodyweight were not significantly different from control and this finding was therefore considered to be coincidental and to reflect normal biological variation. In the low-dose P female group bodyweight-relative brain weight was significantly lower than control. In the absence of any dosage relationship, and any effect on absolute brain weight, this was considered to be incidental and unrelated to treatment (see Table 5 under “Any other information on results incl. tables”).

GROSS PATHOLOGY (PARENTAL ANIMALS)
At 25000 ppm the brain of three P males were observed to be surrounded by clear fluid at necropsy, the brain form one of these animals was also misshapen. One control animal was also observed to have fluid surrounding the brain. In the absence of any evidence of treatment-related histopathological change or any similar findings for P females or for either sex in the F1 generation, this finding was considered to be incidental and unrelated to treatment. The majority of macroscopic necropsy findings of P females were limited to decedent animals; these deaths were considered to be unrelated to treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no histopathological findings observed for adults that were considered to indicate an adverse effect of the test substance at a dietary inclusion level of 25000 ppm.

POSITIVE CONTROL (PARENTAL ANIMALS)
Treatment at 10000 ppm DEPH was associated with clear signs of adult toxicity including notable effects on bodyweight and some organ weights. Reproduction of P animals was unaffected by treatment but shorter male ano-genital distance, increased male nipple counts and delays in attainment of sexual maturation suggested a possible endocrine disruption effect.
Dose descriptor:
NOAEL
Remarks:
toxicity/fertility
Effect level:
>= 25 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Remarks:
toxicity/fertility
Effect level:
>= 1 159 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: mean achieved dose level (P males)
Dose descriptor:
NOAEL
Remarks:
toxicity/fertility
Effect level:
>= 2 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: mean achieved dose level (P females)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
25000 ppm F1: litter weights were slightly lower; 1500/6000 ppm F2: bodyweight gain was slightly lower, non-adverse.
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
1500 ppm F1 males and 6000 ppm F2 females: lower ano-genital distance; 25000 ppm F1 females: visible nipple counts marginally higher, non-adverse.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
25000 ppm female F1/F2: absolute and bodyweight-relative spleen weight decreased; 1500 ppm female F2: lower absolute and bodyweight-relative spleen weights; 6000 ppm female F1: bodyweight-relative uterus weights decreased, non-adverse.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
VIABILITY (OFFSPRING)
Survival of the offspring to weaning was unaffected at the dietary concentrations by either maternal treatment or by more direct treatment when the offspring consumed the diet as they approached weaning in both generations.

CLINICAL SIGNS (OFFSPRING)
No adverse effects were observed for the test substance treated animals.

FOOD INTAKE (OFFSPRING)
Higher food intake for F1 females in the mid-dose group was observed during the last two weeks of gestation and Days 4-7 of lactation. F1 females in the low-dose group showed a significant higher food intake was also observed for the last week of gestation and the first week of lactation. In the absence of any consistent dosage relationship and the absence of any similar increases at the high-dose group, these differences were considered to be unrelated to treatment.

BODY WEIGHT (OFFSPRING)
There was no adverse effect of treatment on litter weights or bodyweight changes. For the F1 offspring at the high-dose litter weights were slightly lower between Days 14-21 (see Table 2 under “Any other information on results incl. tables”). For F2 offspring at the low and high-dose group bodyweight gain was slightly lower than control for both sexes between Day 7-14. These differences may reflect normal biological variation and were not considered to indicate an adverse effect of treatment.

SEXUAL MATURATION (OFFSPRING)
There were no obvious effects of treatment on the sexual maturation of either sex for the F1 animals receiving the test substance. Sex ratio at birth and subsequently at Day 21 of age was similar to concurrent control in all treatment groups over both generations. There was no adverse effect of treatment on mean ano-genital distance on Day 1 of age for either sex. For F1 males at the low-dose and F2 females at the mid-dose, mean ano-genital distance on Day 1 of age was slightly shorter (see Table 4 under “Any other information on results incl. tables”). These decreases were considered to reflect normal biological variation and were considered to be unrelated to treatment.
There was no adverse effect of treatment on visible nipple counts on Days 11-15 of age for either sex in either generation. Only for F1 females at the high-dose visible nipple counts were marginally higher than control only at Day 11 of age, therefore this effect was considered to be incidental and unrelated to treatment (see Table 6 under "Any other information on results incl. tables").

REPRODUCTIVE PERFORMANCE (OFFSPRING)
In total there were 21, 24, 22 and 24 pregnant F1 females at 0 (control), 1500, 6000 and 25000 ppm of the test substance, respectively, that successfully reared young to weaning. One control F1 and one mid-dose F1 female showed total litter loss post partum. As there was no consistency between the F0 and F1 generations and also no increase in mortality for offspring for those litters surviving to weaning in either generation, these isolated occurrences were considered to be incidental and unrelated to maternal treatment.

ORGAN WEIGHTS (OFFSPRING)
For female offspring receiving the high-dose, absolute and bodyweight-relative spleen weights were statistically lower than control in both generations. Statistically lower absolute and bodyweight-relative spleen weights were observed for F2 females at the low-dose. In the absence of any effect on spleen weight or any treatment related histopathological change for adult F1 females, this effect was considered to be of no long term toxicological significance. At the mid-dose, bodyweight-relative uterus weights of F1 offspring were significantly lower than control although absolute weights were not similarly affected in the absence of any effects for F2 offspring at this dosage, or any similar effect at the high-dose, this was considered to be incidental and unrelated to treatment. Assessment of intergroup differences in absolute and bodyweight-relative brain weights at Day 70 of age for offspring derived from dams receiving the test substance did not reveal any obvious adverse effects of previous maternal/pre-weaning exposure.

GROSS PATHOLOGY (OFFSPRING)
The macroscopic abnormalities observed for both decedent and terminal kill offspring were typical for the age examined and neither the incidence or distribution of these findings indicated any adverse effect of treatment with the test substance in either generation.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (OFFSPRING)
One high-dose animal of the F1 generation showed an irregular cycle.

POSITIVE CONTROL (OFFSPRING)
Adverse effects on offspring survival and growth were apparent and offspring failed to thrive following separation from the parent female at weaning. Effects on brain, spleen, thymus and, for females, uterus weight were apparent for the F1 offspring. Necropsy revealed enlarged liver and kidneys, small testes, epididymides and seminal vesicles for male F1 animals.

HISTOPATHOLOGICAL FINDINGS FOR BEHAVIOURAL OFFSPRING
There were no histopathological findings observed for male and female behavioural offspring that were considered to indicate an adverse effect at the high-dose level.
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
>= 25 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
>= 1 342 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: mean achieved dose level (F1 males)
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
>= 2 262 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: mean achieved dose level (F1 females)
Reproductive effects observed:
not specified

Table 1. Overall achieved intakes during each generation for test material treated groups.

Dietary Concentration

Males (mg/kg bw/day)
   F0   F1   

Females (mg/kg bw/day)
Maturation Gestation Lactation


 F0     F1     F0      F1     F0    F1

1500 ppm 82 109 106 135 101 108 231 238
6000 ppm 324 435 431 540 411 434 919 918
25000 ppm 1159 1342 1392 1493 1665 1697 3544 3596

Table 2. Table for reproductive performance.

Parameter

Control group

Low-dose group

1500 ppm

Mid-dose group 6000 ppm

High-dose group 25000 ppm

Live Birth Index

F0-F1

F1-F2

 

99.3

99.6

 

98.2

98.3

 

98.4

99.5

 

98.4

99.5

Pre-Implantation Loss [%]

F0-F1

F1-F2

 

5.4 ± 4.6

5.5 ± 8.7

 

6.8 ± 7.4

4.9 ± 8.8

 

5.7 ± 4.0

5.4 ± 9.8

 

5.4 ± 6.1

1.7 ± 3.1

Post-Implantation Loss [%]

F0-F1

F1-F2

 

3.1 ± 6.5

7.0 ± 8.7

 

3.7 ± 5.5

4.2 ± 5.2

5.0 ± 7.4

4.9 ± 7.8

10.8 ± 16.2**

7.7 ± 13.8

Total Litter Weight (g)

F1 Day 1

F1 Day 14

F1 Day 21

F2 Day 1

F2 Day 14

F2 Day 21

95.1

389.5

600.0

87.3

363.2

564.2

 

89.6

362.6

567.4

101.0

384.1

599.3

 

92.4

368.8

584.8

95.6

368.9

570.4

88.9

343.7**

523.8**

98.0

360.0

555.2

Total number of Corpora Lutea

F0-F1

F1-F2

 

15.7 ± 1.6

15.3 ± 2.3

 

15.7 ± 2.7

17.2 ± 2.2

 

15.7 ± 1.8

16.5 ± 2.9

 

15.9 ± 2.0

16.3 ± 3.8

Total number of Implantation Sites

F0-F1

F1-F2

 

 14.8 ± 1.7

14.4 ± 2.5

 

 

14.5 ± 2.7

16.3 ± 1.8

  

14.8 ± 2.0

15.4 ± 2.4

 

15.0 ± 1.7

16.0 ± 3.7

Total Number of Offspring Born

F0-F1

F1-F2

 

14.4 ± 2.1

13.5 ± 2.9

  

14.0 ± 2.6

15.5 ± 1.7

  

14.2 ± 2.4

14.6 ± 2.5

  

13.4 ± 1.7

14.8 ± 4.0

Sex ratio (% male at birth)

51.9

45.3

46.8

51.8

p** ≤ 0.01

Table 3. Results of sperm characterisation.

Parameter

Control group

 High-dose group 25000 ppm

Sperm concentration [M/mL]

F0

F1 

 

92.2

107.0 

 

102.5

107.0 

Motility (%)

F0

F1 

 

52

58 

 

57

58

Sperm Morphology, Number(%)

F0

F1

 

99.9

100.0

 

99.8

100.0

Table 4. Ano-Genital distance of offspring - group mean litter values.

Ano-genital distance (mm) on Day 1 post partum

Control group

Low-dose group

1500 ppm

Mid-dose group 6000 ppm

High-dose group 25000 ppm

Males

F1

F2

3.12 ± 0.34

3.01 ± 0.33

2.95* ± 0.40

3.03 ± 0.33

3.12 ± 0.46

3.01 ± 0.33

3.13 ± 0.36

3.11 ± 0.31

Females

F1

F2

1.60 ± 0.34

1.47 ± 0.19

1.65 ± 0.44

1.51± 0.24

 

1.49 ± 0.31

1.33 ± 0.14**

 

1.67 ± 0.34

1.49 ± 0.22

p** ≤ 0.01

Table 5. Organ weights.

Absolute organ weights – group mean values

Organ

Control group

Low-dose group

1500 ppm

Mid-dose group

6000 ppm

High-dose group

25000 ppm

Left testes

F0 males

F1 males

 

1.8398 ± 0.2431

1.8445 ± 0.1758

 

1.9042 ± 0.1890

1.8268 ± 0.1048

 

1.9683 ± 0.1463*

1.8508 ± 0.1759

 

1.9484 ± 0.1093

1.8158 ± 0.1950

Thyroid females

F0

F1

 

0.0168 ± 0.0036

0.0191 ± 0.0042

 

0.0170 ± 0.0032

0.0184 ± 0.0053

 

0.0167 ± 0.0036

0.0182 ± 0.0033

 

0.0212± 0.0044***

0.0182 ± 0.0045

Uterus females

F0

F1

 

0.5181 ± 0.1530

0.5396 ± 0.1622

 

0.4434 ± 0.112

0.5563 ± 0.1649

 

0.4640 ± 0.2018*

0.5428 ± 0.1649

 

0.4291 ± 0.0856*

0.5226 ± 0.1183

Organ weights for female offspring – group mean values

Spleen absolute

F1

F2

Spleen. relative

F1

F2

 

0.1861 ± 0.0576

0.1868 ± 0.0646

 

0.4226 ± 0.0615

0.4096 ± 0.0799

 

0.1644 ± 0.0526

0.1451 ± 0.0443*

 

0.3899 ± 0.0879

0.3507 ± 0.0527*

 

0.1642 ± 0.0589

0.1534 ± 0.0493

 

0.3762 ± 0.0764

0.3656 ± 0.0713

 

0.1485 ± 0.0513*

0.1370 ± 0.0590**

 

0.3553 ± 0.0719**

0.3257 ± 0.0748***

Uterus absolute

F1

F2

Uterus relative

F1

F2

 

0.0923 ± 0.0265

0.0539 ± 0.0113

 

0.2111 ± 0.0322

0.1210 ± 0.0158

 

0.0877 ± 0.0265

0.0536 ± 0.0182

 

0.2119 ± 0.0601

0.1324 ± 0.0426

 

0.0831 ± 0.0274

0.0513 ± 0.0212

 

0.1931 ± 0.0456*

0.1225 ± 0.0348

 

0.1242 ± 0.1746

0.0517 ± 0.0242

 

0.2967 ± 0.4169

0.1276 ± 0.0489

p* ≤ 0.05, p** ≤ 0.01, p*** ≤ 0.001

Table 6. Nipple counts for the F1 generation at Day 11 of age.

Group

Mean Nipple count at Day 11 of age

Male

Female

Control

0.0 ± 0.0

9.4 ± 1.8

Low dose group

0.1 ± 0.5

10.4 ± 1.5

Mid-dose group

0.0 ± 0.0

9.9 ± 1.5

High-dose group

0.1 ± 0.3

10.6 ± 1.4*

p* ≤ 0.05

Conclusions:
The test substance had no effect on reproductive performance.
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Feb - 07 April 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP-Guideline study, tested with the source substance glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS 91052-13-0). According to the ECHA guidance document ‘Practical guide 6: How to report read-across and categories' (ECHA, 2012), the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France
- Age at study initiation: approximately 12 weeks
- Weight at study initiation: 277-353 g (males), 180-228 g (females)
- Housing : 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). This was also applicable for Main females and Recovery animals throughout the complete treatment period. Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 (actual range: 19.7-21.9)
- Humidity (%): 40-70 (actual range: 22-71)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. The testing laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6h prior to dosing and were homogenized to a visually acceptable level.
Adjustment was made for the density of the test substance (0.944), and the specific gravity of the vehicle (1.125).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Purity: polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany)

Dose volume: 5 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or by the appearance of an intravaginal copulatory plug referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type).

Following a minimum of 14 days of exposure for the males and females, one Repro female was cohabitated with one Main male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates). Detection of mating was not confirmed for animal no. 98 which did deliver. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating. After 14 days of mating, females who had not shown evidence of mating were separated from their males.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability. Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6h at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤10%) and formulations at the entire range were stable when stored at room temperature for at least 6h.
Duration of treatment / exposure:
41-49 days,
i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.
Frequency of treatment:
daily, 7 days/week
Details on study schedule:
Offspring were euthanized at the age of 4 days.
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 10-day dose range finding study
Parental animals: Observations and examinations:
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded.
Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes:

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND: Yes

for further details see Section 7.5.1
Oestrous cyclicity (parental animals):
Uterus epithelium was analysed histologically for estrus, proestrus and cystic endometrial glands.
Sperm parameters (parental animals):
Parameters examined in [all/P/F1/F2] male parental generations: testis weight, epididymis weight, histology of testes
Of the first 5 Main males of the control and high dose group, additional slides of the testes were prepared to examine staging of spermatogenesis.
The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin.
Litter observations:
On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.
Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made in all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.
Postmortem examinations (parental animals):
SACRIFICE
- Maternal animals which delivered: on lactation Day 5
- Maternal animals which failed to deliver (4 animals): Post-coitum Day 26 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).

GROSS NECROPSY
- Gross necropsy consisted of: see details under 7.5.1
The numbers of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY / ORGAN WEIGHTS
see details under 7.5.1
Postmortem examinations (offspring):
SACRIFICE
- Pups surviving to planned termination were killed by decapitation on lactation Day 5.

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue as well as pups from females that were killed in extremis were preserved in 10% buffered formalin for possible further examination.
Statistics:
See Section 7.5.1
Reproductive indices:
For each group the following calculations were performed:
- Mating (%): Number of females mated/Number of females paired x 100
- Fertility index (%): Number of pregnant females/Number of females paired x 100
- Conception index (%): Number of pregnant females/Number of females mated x 100
- Gestation index (%): Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100
Offspring viability indices:
For each group the following calculations were performed:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
as found by histological examination of uterus
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
as found by histological examination of testes
Reproductive performance:
no effects observed
REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis, as explored by histological examination of testes.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Histological examination of the uterus epithelium and endometrial glands did not reveal any treatment related influences on estrous cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No treatment related effects on reproductive parameters were noted.
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No treatment-related effects observed.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects observed.

For further details see 7.5.1
Dose descriptor:
NOAEL
Remarks:
parental fertility
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Number of pups: 432
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed at the doses up to 1000 mg/kg bw/day
Reproductive effects observed:
not specified
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
refer to category justification report provided in IUCLID section 13.
Reason / purpose:
read-across source
Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France
- Age at study initiation: approximately 12 weeks
- Weight at study initiation: 277-353 g (males), 180-228 g (females)
- Housing : 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). This was also applicable for Main females and Recovery animals throughout the complete treatment period. Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 (actual range: 19.7-21.9)
- Humidity (%): 40-70 (actual range: 22-71)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. The testing laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6h prior to dosing and were homogenized to a visually acceptable level.
Adjustment was made for the density of the test substance (0.944), and the specific gravity of the vehicle (1.125).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Purity: polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany)

Dose volume: 5 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or by the appearance of an intravaginal copulatory plug referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type).

Following a minimum of 14 days of exposure for the males and females, one Repro female was cohabitated with one Main male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates). Detection of mating was not confirmed for animal no. 98 which did deliver. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating. After 14 days of mating, females who had not shown evidence of mating were separated from their males.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability. Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6h at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤10%) and formulations at the entire range were stable when stored at room temperature for at least 6h.
Duration of treatment / exposure:
41-49 days,
i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.
Frequency of treatment:
daily, 7 days/week
Details on study schedule:
Offspring were euthanized at the age of 4 days.
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 10-day dose range finding study
Parental animals: Observations and examinations:
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded.
Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes:

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND: Yes

for further details see Section 7.5.1
Oestrous cyclicity (parental animals):
Uterus epithelium was analysed histologically for estrus, proestrus and cystic endometrial glands.
Sperm parameters (parental animals):
Parameters examined in [all/P/F1/F2] male parental generations: testis weight, epididymis weight, histology of testes
Of the first 5 Main males of the control and high dose group, additional slides of the testes were prepared to examine staging of spermatogenesis.
The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin.
Litter observations:
On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.
Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made in all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.
Postmortem examinations (parental animals):
SACRIFICE
- Maternal animals which delivered: on lactation Day 5
- Maternal animals which failed to deliver (4 animals): Post-coitum Day 26 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).

GROSS NECROPSY
- Gross necropsy consisted of: see details under 7.5.1
The numbers of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY / ORGAN WEIGHTS
see details under 7.5.1
Postmortem examinations (offspring):
SACRIFICE
- Pups surviving to planned termination were killed by decapitation on lactation Day 5.

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue as well as pups from females that were killed in extremis were preserved in 10% buffered formalin for possible further examination.
Statistics:
See Section 7.5.1
Reproductive indices:
For each group the following calculations were performed:
- Mating (%): Number of females mated/Number of females paired x 100
- Fertility index (%): Number of pregnant females/Number of females paired x 100
- Conception index (%): Number of pregnant females/Number of females mated x 100
- Gestation index (%): Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100
Offspring viability indices:
For each group the following calculations were performed:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
as found by histological examination of uterus
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
as found by histological examination of testes
Reproductive performance:
no effects observed
REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis, as explored by histological examination of testes.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Histological examination of the uterus epithelium and endometrial glands did not reveal any treatment related influences on estrous cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No treatment related effects on reproductive parameters were noted.
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No treatment-related effects observed.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects observed.

For further details see 7.5.1
Dose descriptor:
NOAEL
Remarks:
parental fertility
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Number of pups: 432
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed at the doses up to 1000 mg/kg bw/day
Reproductive effects observed:
not specified
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Reason / purpose:
read-across source
Species:
rat
Strain:
other: Sprague-Dawley Crl:CD® (SD) IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, UK
- Age at study initiation: (P) 7-8 weeks
- Weight at study initiation: (P) 216-361 g (males) and 149-249 g (females)
- Housing: All P animals were housed in groups of up to 4 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper. Mated females were housed individually (or with their litter), in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes (Datesand Ltd., Cheshire, UK). For the offspring selected to form the F1 generation, this housing procedure was repeated. The P and F1 offspring selected for assessment of developmental neurotoxicity were housed in groups of up to 4 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding.
- Diet: ground diet (Rodent PMI 5002 Diet, BCM IPS Limited, London, UK), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Each concentration of the test material was prepared by weighing an appropriate amount and mixing it with a small amount of the required volume of diet. Once this was adequately mixed the formulation was then transferred to a Hobart H800 mixer and mixed with the remaining required volume of diet.

DIET PREPARATION
- Mixing appropriate amounts with: laboratory diet

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating.
- After 14 days of unsuccessful pairing replacement of first male by another male or extension of the mating phase to a 3 week.
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test material in the dietary admixtures was determined by gas chromatography using an external standard technique. The dietary admixtures were sampled and analysed within 2 weeks of preparation. Homogeneity and stability determinations were performed under Harlan Laboratories Ltd. project number 2384/0006. The results showed that achieved concentrations were between 95 and 121% of nominal concentration.
Duration of treatment / exposure:
(P) Males: 10 weeks during maturation and throughout mating, gestation and until completion of the P female lactation phase.
(P) Females: 10 weeks during maturation and throughout mating, gestation and until Day 21 lactation phases.
(F1) Females: minimum of 10 weeks during maturation and subsequently throughout mating, gestation and lactation phases. Between weaning and the formal start of the F1 generation, the animals continued to receive their appropriate treated diet.
(F1) Males: minimum of 10 weeks during maturation and subsequently throughout mating, gestation and until completion of the F1 female lactation phases. Between weaning and the formal start of the F1 generation, the animals continued to receive their appropriate treated diet.
(F2) Males/ Females: from weaning (Day 21 of age) to termination at Day 70 of age.
Frequency of treatment:
daily, 7 days/week
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 10 weeks
- Due to number of offspring required for developmental neurotoxicity, the study was split into two equal parts. Each group was divided into two equal sub-groups; with animals allocated to the second part being given treated diets eight days later. Chronically, all procedures occurred approximately one week later for the second sub-group compared to the first group.
Remarks:
Doses / Concentrations:
1500, 6000 and 25000 ppm
Basis:
other: nominal in diet: animals in the high dose group initially received 15000 ppm rising to 20000 ppm to 25000 ppm during the maturation phase of each generation
Remarks:
Doses / Concentrations:
82, 324 and 1159 mg/kg bw/day
Basis:
other: mean achieved dose level (P males)
Remarks:
Doses / Concentrations:
146, 587 and 2200 mg/kg bw/day
Basis:
other: mean achieved dose level (P females)
Remarks:
Doses / Concentrations:
109, 435 and 1342 mg/kg bw/day
Basis:
other: mean achieved dose level (F1 males)
Remarks:
Doses / Concentrations:
160, 630 and 2262 mg/kg bw/day
Basis:
other: mean achieved dose level (F1 females)
No. of animals per sex per dose:
28 P males, 28 P females
24 F1 males, 24 F1 females
Control animals:
other: yes, laboratory diet treated with Arachis oil to ensure comparable calorific intake
Details on study design:
- Dose selection rationale: The dietary levels were based on known toxicology data including an earlier preliminary study in the rat (Project number 2384/0006).
- Other: In each generation, animals receiving the high dose level initially received the test material at a concentration of 15000 ppm and this was increased to 20000 ppm and finally 25000 ppm as the study progressed. Animals were receiving the highest inclusion level by the end of the maturation/pre-pairing phase in both generations and therefore were receiving the higher dietary inclusion by the time of the main reproductive assessment.
Positive control:
The study incorporated a positive control for endocrine disruption, DEHP (bis(2-thylhexyl) phthalate), throughout the P generation and until sexual maturation of the F1 ofspring. Animals received an intended dietary inclusion level of 10000 ppm.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the week and once daily on weekends and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1, prior to treatment, weekly throughout the maturation phase of each generation and continuing for males until termination. Following pairing P/F1 females were weighted daily until mating was evident. Mated females were weighted on Day 0, 7, 14 and 21 post coitum and for females that littered on Day 1, 4, 7, 14 and 21 post partum. P/F1 offspring (selected for post-weaning assessment of developmental neurotoxicity) were weighted on Day 28, 35, 42, 49, 56, 63 and 70 of age. Body weight was recorded for all animals on the Day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption was assessed for each cage of adults during the maturation period and for males after mating/pairing. Females showing evidence of mating, food consumption was recorded for the periods Day 0-7, 7-14 and 14-21 post coitum. For females that littered, food consumption was recorded for the period covering Day 1-4, 4-7, 7-14 and 14-21 post partum.

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: daily by visual inspection for any overt change
Oestrous cyclicity (parental animals):
Prior to pairing of females for the P and F1 mating phases, a vaginal smear was taken daily for twenty-one days and a sample was placed on a glass slide. The smears were allowed to dry and then stained using a diluted giemsa stain and examined microscopically.
Sperm parameters (parental animals):
Parameters examined in P/F1 male parental generations in control and high-dose males: Yes. Testis weight, epididymis weight, spermatid enumeration, sperm motility, sperm morphology.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in P/ F1 offspring: number and sex of pups, live births, postnatal mortality reported on Day 1, 4, 7, 14 and 21, clinical condition, individual bodyweight, necropsy findings, ano-genital distance and number of visible nipples on Day 11 and 15 and neurobehavioural effects.

GROSS EXAMINATION OF DEAD PUPS:
Yes, dead offspring was subjected to a necropsy examination.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving male F0/F1 animals were killed following completion of the F0 female lactation phase.
- Maternal animals: All surviving F0/F1 females were killed and examined macroscopically at Day 21 of lactation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Selected organs (adrenals, coagulating gland, right epididymis, ovaries, right testis, pituitary, prostate, seminal vesicles, uterus with oviducts and cervix, vagina, gross lesions) were weighed and/or preserved for histopathological examination.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- The P and F1 offspring selected for the post-weaning-developmental neurotoxicity were killed at Day 70 of age.
- All unselected offspring and those dying during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS
The following tissues were preserved from all F1 males and females from each dose group, in buffered 10% formalin: adrenals, coagulating gland, right epididymis, ovaries, right testis, pituitary, prostate, seminal vesicles, uterus with oviducts and cervix, vagina, gross lesions. The tissues from all control and high-dose animals and any treated animals which failed to mate or that did not achieve a pregnancy were processed. In addition, the following tissues were prepared for microscopic examination and weighed, respectively. The brain, spleen, thymus and uterus were weighed and preserved for one unselected male/female F1/F2 offspring at necropsy. In addition, samples of the following tissues of perfused animals at Day 70 of age (selected for the post-weaning-developmental neurotoxicity examinations) were histopathologically investigated: brain, dorsal root ganglia, dorsal and ventral root fibres, eyes, optic nerve, peroneal nerve, sciatic nerve, sural nerve, tibial nerve, skeletal muscle and spinal cord.
Statistics:
Data were initially assessed for homogeneity of variance using Levene´s test. Where variances were shown to be homogenous, a parametric assessment of the data was performed using one way analysis of variance (ANOVA), which if significant was followed by pairwise comparisons using Dunnett´s test. Where Levene´s test showed unequal variances, the data were analysed using non-parametric methodology: Kruskal-Wallis ANOVA which, if significant, was followed by Mann-Whitney U-test. Probability values (p) are presented as follows: p ≤ 0.001***, p ≤ 0.01**, p ≤ 0.05*, p > 0.05 (not significant).
Reproductive indices:
Mating performance and fertility
- Pre-coital interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

Fertility Indices
- Mating Index (%): (Number of animals mated/ number of animals paired) x 100
- Pregnancy Index (%): (Number of pregnant females/ number of females mated) x 100

Gestation and Parturition Data
- Gestation length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition. Where the start of parturition occurred overnight, the total was adjusted by adding half a day.
- Parturition Index (%): (Number of females delivering live offspring/ number of pregnant females) x 100

- Sex Ratio (% males): (Number of male offspring/ total number of offspring) x 100
Offspring viability indices:
Lactation Data
- Implantation losses (%):
% pre-implantation loss = [(Number of corpora lutea - number of implantation sites) / number of corpora lutea] x 100
% post-implantation loss = [(Group number of implantation sites - number of offspring) / number of implantation sites] x 100
offspring) x 100

Live Birth and Viability Indices
- Live Birth Index (%) = (Number of offspring alive on Day 1/ number of offspring born) x 100
- Viability Index 1 (%) = (Number of offspring alive on Day 4/ number of offspring alive on Day 1) x 100
- Viability Index 2 (%) = (Number of offspring alive on Day 7/ number of offspring alive on Day 4) x 100
- Viability Index 3 (%) = (Number of offspring alive on Day 14/ number of offspring alive on Day 7) x 100
- Viability Index 4 (%) = (Number of offspring alive on Day 21/ number of offspring alive on Day 14) x 100
- Viability Index 5 (%) = (Number of offspring alive on Day 21/ number of offspring alive on Day 1) x 100

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
25000 ppm: one P female was killed due to a decline in clinical condition; control/6000 ppm: one P female in each group was killed due to adverse clinical condition around the time of expected parturition, not treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1500/25000 ppm: increase in bodyweight gain during Days 1-21; 25000 ppm (P, female): significant lower food intake during the last week of lactation; 6000 ppm (P, female): higher food intake during last week of gestation and lactation, non-adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1500/25000 ppm: increase in bodyweight gain during Days 1-21; 25000 ppm (P, female): significant lower food intake during the last week of lactation; 6000 ppm (P, female): higher food intake during last week of gestation and lactation, non-adverse.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
6000 ppm/control: one P female animal of each group showed an irregular cycle, non-adverse.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
25000 ppm: lower homogenisation resistant spermatid counts (P amles) for the epididymides and testes, non-adverse.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
25000 ppm: lower number of P females with live offspring, higher post-implantation loss, not treatment-related.
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no treatment-related deaths amongst parental animals. One female in the high-dose group showed dehydration, lethargy, emaciation, hunched posture, tip-toed gait and staining around mouth. Macroscopic necropsy examination indicated that the animal had a suspected broken jaw and the decline in clinical condition was considered to be unrelated to treatment. One control P female showed hunched posture, pilo-erection, tip-toed gait, lethargy pallor of the extremities and vaginal discharge on Day 97 of the P generation. One female in the mid-dose group showed hunched posture, pilo-erection, staining around the snout, staining around ano-genital region and pallor of the extremities on Day 99 of the P generation. These clinical signs coincided with the time of expected parturition and were considered to reflect difficulties with the birth of their litter. These animals were therefore killed for animal welfare considerations.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No adverse effects of treatment on mean bodyweight or bodyweight change was apparent in either generation for males and females. A significant increase in bodyweight gain during Days 1-21 in the low- and high-dose female P group was considered to be not treatment related.
No adverse effect of treatment on mean food consumption was apparent in males and females. Food consumption of P females in the high-dose group was significantly lower during the last week of lactation. In mid-dose P females a higher food intake was observed during the last week of gestation and lactation.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Achieved intakes for animals were in line with expectations throughout the P and F1 generations (see Table 1 under “Any other information on results incl. tables”).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no obvious adverse effects of treatment on the oestrous cycles of females. One control and one mid-dose animal of the P generation showed an irregular cycle. There was no adverse effect of treatment on corpora lutea count, numbers of implantation sites or litter size at birth for females receiving the test substance. The mean number of small, medium or large oocyte follicles in the ovaries for F1 females of the high-dose group did not indicate any adverse effects of treatment (see Table 2 under “Any other information on results incl. tables”).

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No adverse effects of treatment on sperm concentration, motility or morphology were apparent for males receiving the test substance in either generation. For P males at the high-dose, homogenisation resistant spermatid counts for the epididymides and testes were lower than concurrent control, this decrease was considered to be incidental and of no toxicological significance (see Table 3 under “Any other information on results incl. tables”).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no obvious adverse effects of treatment on mating performance, fertility or gestation length of females receiving the test substance. While the number of P females with live offspring was marginally lower than anticipated in the high-dose group, there was no similar occurrence in the following F1 generation and this finding was therefore considered coincidental and unrelated to treatment.
In total there were 26, 25, 26 and 23 P females at 0 (control), 1500, 6000 and 25000 ppm of the test substance, respectively, that successfully reared young to weaning. In P females at the high-dose group a higher post-implantation loss was observed (see Table 3 under “Any other information on results incl. tables”). There was no subsequent significant difference in litter size at birth for the P females at the high-dose and no similar increase in post-implantation loss for F1 females at this dosage. In view of this, the higher implantation loss observed was considered to be incidental and unrelated to treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
For P males receiving the mid-dose, absolute and bodyweight-relative organ weight for the left testis was significantly higher than control. In the absence of any significant increase for the right testis for these males or both testes for F1 males or any treatment related histopathological change, this effect was considered to be incidental and unrelated to treatment. For P females of the high-dose group, absolute and bodyweight-relative thyroid weights were significantly higher. There were no significant differences in thyroid weights for F1 females or for males in either generation at this inclusion level. Given the earlier and potentially higher exposure of the F1 females to the test substance (due to low bodyweight at this stage) and the absence of any accompanying effect on thyroid weights for these animals, this finding was therefore considered to be incidental and of no toxicological significance. For P females receiving the mid and high-dose, absolute uterine weight was significantly lower; values when adjusted for the bodyweight were not significantly different from control and this finding was therefore considered to be coincidental and to reflect normal biological variation. In the low-dose P female group bodyweight-relative brain weight was significantly lower than control. In the absence of any dosage relationship, and any effect on absolute brain weight, this was considered to be incidental and unrelated to treatment (see Table 5 under “Any other information on results incl. tables”).

GROSS PATHOLOGY (PARENTAL ANIMALS)
At 25000 ppm the brain of three P males were observed to be surrounded by clear fluid at necropsy, the brain form one of these animals was also misshapen. One control animal was also observed to have fluid surrounding the brain. In the absence of any evidence of treatment-related histopathological change or any similar findings for P females or for either sex in the F1 generation, this finding was considered to be incidental and unrelated to treatment. The majority of macroscopic necropsy findings of P females were limited to decedent animals; these deaths were considered to be unrelated to treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no histopathological findings observed for adults that were considered to indicate an adverse effect of the test substance at a dietary inclusion level of 25000 ppm.

POSITIVE CONTROL (PARENTAL ANIMALS)
Treatment at 10000 ppm DEPH was associated with clear signs of adult toxicity including notable effects on bodyweight and some organ weights. Reproduction of P animals was unaffected by treatment but shorter male ano-genital distance, increased male nipple counts and delays in attainment of sexual maturation suggested a possible endocrine disruption effect.
Dose descriptor:
NOAEL
Remarks:
toxicity/fertility
Effect level:
>= 25 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Remarks:
toxicity/fertility
Effect level:
>= 1 159 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: mean achieved dose level (P males)
Dose descriptor:
NOAEL
Remarks:
toxicity/fertility
Effect level:
>= 2 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: mean achieved dose level (P females)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
25000 ppm F1: litter weights were slightly lower; 1500/6000 ppm F2: bodyweight gain was slightly lower, non-adverse.
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
1500 ppm F1 males and 6000 ppm F2 females: lower ano-genital distance; 25000 ppm F1 females: visible nipple counts marginally higher, non-adverse.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
25000 ppm female F1/F2: absolute and bodyweight-relative spleen weight decreased; 1500 ppm female F2: lower absolute and bodyweight-relative spleen weights; 6000 ppm female F1: bodyweight-relative uterus weights decreased, non-adverse.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
VIABILITY (OFFSPRING)
Survival of the offspring to weaning was unaffected at the dietary concentrations by either maternal treatment or by more direct treatment when the offspring consumed the diet as they approached weaning in both generations.

CLINICAL SIGNS (OFFSPRING)
No adverse effects were observed for the test substance treated animals.

FOOD INTAKE (OFFSPRING)
Higher food intake for F1 females in the mid-dose group was observed during the last two weeks of gestation and Days 4-7 of lactation. F1 females in the low-dose group showed a significant higher food intake was also observed for the last week of gestation and the first week of lactation. In the absence of any consistent dosage relationship and the absence of any similar increases at the high-dose group, these differences were considered to be unrelated to treatment.

BODY WEIGHT (OFFSPRING)
There was no adverse effect of treatment on litter weights or bodyweight changes. For the F1 offspring at the high-dose litter weights were slightly lower between Days 14-21 (see Table 2 under “Any other information on results incl. tables”). For F2 offspring at the low and high-dose group bodyweight gain was slightly lower than control for both sexes between Day 7-14. These differences may reflect normal biological variation and were not considered to indicate an adverse effect of treatment.

SEXUAL MATURATION (OFFSPRING)
There were no obvious effects of treatment on the sexual maturation of either sex for the F1 animals receiving the test substance. Sex ratio at birth and subsequently at Day 21 of age was similar to concurrent control in all treatment groups over both generations. There was no adverse effect of treatment on mean ano-genital distance on Day 1 of age for either sex. For F1 males at the low-dose and F2 females at the mid-dose, mean ano-genital distance on Day 1 of age was slightly shorter (see Table 4 under “Any other information on results incl. tables”). These decreases were considered to reflect normal biological variation and were considered to be unrelated to treatment.
There was no adverse effect of treatment on visible nipple counts on Days 11-15 of age for either sex in either generation. Only for F1 females at the high-dose visible nipple counts were marginally higher than control only at Day 11 of age, therefore this effect was considered to be incidental and unrelated to treatment (see Table 6 under "Any other information on results incl. tables").

REPRODUCTIVE PERFORMANCE (OFFSPRING)
In total there were 21, 24, 22 and 24 pregnant F1 females at 0 (control), 1500, 6000 and 25000 ppm of the test substance, respectively, that successfully reared young to weaning. One control F1 and one mid-dose F1 female showed total litter loss post partum. As there was no consistency between the F0 and F1 generations and also no increase in mortality for offspring for those litters surviving to weaning in either generation, these isolated occurrences were considered to be incidental and unrelated to maternal treatment.

ORGAN WEIGHTS (OFFSPRING)
For female offspring receiving the high-dose, absolute and bodyweight-relative spleen weights were statistically lower than control in both generations. Statistically lower absolute and bodyweight-relative spleen weights were observed for F2 females at the low-dose. In the absence of any effect on spleen weight or any treatment related histopathological change for adult F1 females, this effect was considered to be of no long term toxicological significance. At the mid-dose, bodyweight-relative uterus weights of F1 offspring were significantly lower than control although absolute weights were not similarly affected in the absence of any effects for F2 offspring at this dosage, or any similar effect at the high-dose, this was considered to be incidental and unrelated to treatment. Assessment of intergroup differences in absolute and bodyweight-relative brain weights at Day 70 of age for offspring derived from dams receiving the test substance did not reveal any obvious adverse effects of previous maternal/pre-weaning exposure.

GROSS PATHOLOGY (OFFSPRING)
The macroscopic abnormalities observed for both decedent and terminal kill offspring were typical for the age examined and neither the incidence or distribution of these findings indicated any adverse effect of treatment with the test substance in either generation.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (OFFSPRING)
One high-dose animal of the F1 generation showed an irregular cycle.

POSITIVE CONTROL (OFFSPRING)
Adverse effects on offspring survival and growth were apparent and offspring failed to thrive following separation from the parent female at weaning. Effects on brain, spleen, thymus and, for females, uterus weight were apparent for the F1 offspring. Necropsy revealed enlarged liver and kidneys, small testes, epididymides and seminal vesicles for male F1 animals.

HISTOPATHOLOGICAL FINDINGS FOR BEHAVIOURAL OFFSPRING
There were no histopathological findings observed for male and female behavioural offspring that were considered to indicate an adverse effect at the high-dose level.
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
>= 25 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
>= 1 342 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: mean achieved dose level (F1 males)
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
>= 2 262 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: mean achieved dose level (F1 females)
Reproductive effects observed:
not specified

Table 1. Overall achieved intakes during each generation for test material treated groups.

Dietary Concentration

Males (mg/kg bw/day)
   F0   F1   

Females (mg/kg bw/day)
Maturation Gestation Lactation


 F0     F1     F0      F1     F0    F1

1500 ppm 82 109 106 135 101 108 231 238
6000 ppm 324 435 431 540 411 434 919 918
25000 ppm 1159 1342 1392 1493 1665 1697 3544 3596

Table 2. Table for reproductive performance.

Parameter

Control group

Low-dose group

1500 ppm

Mid-dose group 6000 ppm

High-dose group 25000 ppm

Live Birth Index

F0-F1

F1-F2

 

99.3

99.6

 

98.2

98.3

 

98.4

99.5

 

98.4

99.5

Pre-Implantation Loss [%]

F0-F1

F1-F2

 

5.4 ± 4.6

5.5 ± 8.7

 

6.8 ± 7.4

4.9 ± 8.8

 

5.7 ± 4.0

5.4 ± 9.8

 

5.4 ± 6.1

1.7 ± 3.1

Post-Implantation Loss [%]

F0-F1

F1-F2

 

3.1 ± 6.5

7.0 ± 8.7

 

3.7 ± 5.5

4.2 ± 5.2

5.0 ± 7.4

4.9 ± 7.8

10.8 ± 16.2**

7.7 ± 13.8

Total Litter Weight (g)

F1 Day 1

F1 Day 14

F1 Day 21

F2 Day 1

F2 Day 14

F2 Day 21

95.1

389.5

600.0

87.3

363.2

564.2

 

89.6

362.6

567.4

101.0

384.1

599.3

 

92.4

368.8

584.8

95.6

368.9

570.4

88.9

343.7**

523.8**

98.0

360.0

555.2

Total number of Corpora Lutea

F0-F1

F1-F2

 

15.7 ± 1.6

15.3 ± 2.3

 

15.7 ± 2.7

17.2 ± 2.2

 

15.7 ± 1.8

16.5 ± 2.9

 

15.9 ± 2.0

16.3 ± 3.8

Total number of Implantation Sites

F0-F1

F1-F2

 

 14.8 ± 1.7

14.4 ± 2.5

 

 

14.5 ± 2.7

16.3 ± 1.8

  

14.8 ± 2.0

15.4 ± 2.4

 

15.0 ± 1.7

16.0 ± 3.7

Total Number of Offspring Born

F0-F1

F1-F2

 

14.4 ± 2.1

13.5 ± 2.9

  

14.0 ± 2.6

15.5 ± 1.7

  

14.2 ± 2.4

14.6 ± 2.5

  

13.4 ± 1.7

14.8 ± 4.0

Sex ratio (% male at birth)

51.9

45.3

46.8

51.8

p** ≤ 0.01

Table 3. Results of sperm characterisation.

Parameter

Control group

 High-dose group 25000 ppm

Sperm concentration [M/mL]

F0

F1 

 

92.2

107.0 

 

102.5

107.0 

Motility (%)

F0

F1 

 

52

58 

 

57

58

Sperm Morphology, Number(%)

F0

F1

 

99.9

100.0

 

99.8

100.0

Table 4. Ano-Genital distance of offspring - group mean litter values.

Ano-genital distance (mm) on Day 1 post partum

Control group

Low-dose group

1500 ppm

Mid-dose group 6000 ppm

High-dose group 25000 ppm

Males

F1

F2

3.12 ± 0.34

3.01 ± 0.33

2.95* ± 0.40

3.03 ± 0.33

3.12 ± 0.46

3.01 ± 0.33

3.13 ± 0.36

3.11 ± 0.31

Females

F1

F2

1.60 ± 0.34

1.47 ± 0.19

1.65 ± 0.44

1.51± 0.24

 

1.49 ± 0.31

1.33 ± 0.14**

 

1.67 ± 0.34

1.49 ± 0.22

p** ≤ 0.01

Table 5. Organ weights.

Absolute organ weights – group mean values

Organ

Control group

Low-dose group

1500 ppm

Mid-dose group

6000 ppm

High-dose group

25000 ppm

Left testes

F0 males

F1 males

 

1.8398 ± 0.2431

1.8445 ± 0.1758

 

1.9042 ± 0.1890

1.8268 ± 0.1048

 

1.9683 ± 0.1463*

1.8508 ± 0.1759

 

1.9484 ± 0.1093

1.8158 ± 0.1950

Thyroid females

F0

F1

 

0.0168 ± 0.0036

0.0191 ± 0.0042

 

0.0170 ± 0.0032

0.0184 ± 0.0053

 

0.0167 ± 0.0036

0.0182 ± 0.0033

 

0.0212± 0.0044***

0.0182 ± 0.0045

Uterus females

F0

F1

 

0.5181 ± 0.1530

0.5396 ± 0.1622

 

0.4434 ± 0.112

0.5563 ± 0.1649

 

0.4640 ± 0.2018*

0.5428 ± 0.1649

 

0.4291 ± 0.0856*

0.5226 ± 0.1183

Organ weights for female offspring – group mean values

Spleen absolute

F1

F2

Spleen. relative

F1

F2

 

0.1861 ± 0.0576

0.1868 ± 0.0646

 

0.4226 ± 0.0615

0.4096 ± 0.0799

 

0.1644 ± 0.0526

0.1451 ± 0.0443*

 

0.3899 ± 0.0879

0.3507 ± 0.0527*

 

0.1642 ± 0.0589

0.1534 ± 0.0493

 

0.3762 ± 0.0764

0.3656 ± 0.0713

 

0.1485 ± 0.0513*

0.1370 ± 0.0590**

 

0.3553 ± 0.0719**

0.3257 ± 0.0748***

Uterus absolute

F1

F2

Uterus relative

F1

F2

 

0.0923 ± 0.0265

0.0539 ± 0.0113

 

0.2111 ± 0.0322

0.1210 ± 0.0158

 

0.0877 ± 0.0265

0.0536 ± 0.0182

 

0.2119 ± 0.0601

0.1324 ± 0.0426

 

0.0831 ± 0.0274

0.0513 ± 0.0212

 

0.1931 ± 0.0456*

0.1225 ± 0.0348

 

0.1242 ± 0.1746

0.0517 ± 0.0242

 

0.2967 ± 0.4169

0.1276 ± 0.0489

p* ≤ 0.05, p** ≤ 0.01, p*** ≤ 0.001

Table 6. Nipple counts for the F1 generation at Day 11 of age.

Group

Mean Nipple count at Day 11 of age

Male

Female

Control

0.0 ± 0.0

9.4 ± 1.8

Low dose group

0.1 ± 0.5

10.4 ± 1.5

Mid-dose group

0.0 ± 0.0

9.9 ± 1.5

High-dose group

0.1 ± 0.3

10.6 ± 1.4*

p* ≤ 0.05

Conclusions:
The test substance had no effect on reproductive performance.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to the endpoint discussion for further details). Taken together, the information from these independent sources is consistent and provides sufficient weight of evidence for hazard assessment leading to an endpoint conclusion in accordance with Annex XI, 1.2, of Regulation (EC) No 1907/2006. Therefore, the available information as a whole is sufficient to fulfil the standard information requirements set out in Annex VIII-X, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for read-across

There are no data on the reproduction toxicity of glycerides, C16-18 and C18-unsaturated mono-and di-citrates (CAS 91052-16-3). The assessment was therefore based on studies conducted with analogue substances as part of a read-across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

CAS No. 91052-13-0

An oral gavage screening toxicity study was performed according to OECD guideline 422 and under conditions of GLP in Crl:WI(Han) Wistar rats at doses of 0, 100, 300 and 1000 mg/kg bw/day (Otterdijk, 2010). Dilutions of the test substance in polyethylene glycol were administered once daily to groups of 10 male and 5 female rats (main animals) via gavage. A similar constituted group received the vehicle and served as a control. In addition, satellite groups of 5 males and 5 females (recovery animals) each for the control and high dose group were used to investigate reversibility of effects during a 14-day post-exposure recovery period. Furthermore, 10 females (repro animals) were added to each group for the assessment of reproduction and developmental toxicity. Main and recovery animals were exposed for at least 28 days from start of treatment up to termination or start of recovery. Females used for the assessment of reproduction/developmental toxicity were exposed for 41-49 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. In parental animals, no effects on reproductive function (spermatogenetic and oestrus cycle) and performance (mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites) were observed after treatment compared to controls. Testis weight, epididymis weight, and histology of testes in males as well as histology of uterus epithelium in female did not reveal any substance-related effects in the parental animals. No toxicologically relevant alterations in offspring viability indices were observed. Therefore, a NOAEL for parental fertility of≥1000 mg/kg bw/day was derived for male and female Crl:WI(Han) Wistar rats.

CAS No. 736150-63-3

A Two-generation reproduction toxicity and developmental neurotoxicity study with Glycerides, castor-oil, mono, hydrogenated, acetates was performed in Sprague-Dawley Crl:CD®(SD) IGS BR rats according to OECD guidelines 416 and 426 and in compliance with GLP (Fulcher, 2011). Groups of 28 parental animals per sex were exposed daily to the test substance at dietary concentrations of 1500, 6000 and 25000 ppm, corresponding to mean achieved dose levels of 82, 324 and 1159 mg/kg bw/day in males and mean achieved dose levels of 146, 587 and 2200 mg/kg bw/day in females, respectively. Parental males were treated with the test substance 10 weeks during maturation and throughout mating, gestation and until completion of the parental female lactation phase. Parental females received the test substance 10 weeks during maturation and throughout mating, gestation and until Day 21 of the lactation phases. After weaning, groups of 24 animals per sex of the F1 generation were exposed daily to the same dietary concentrations as their parental animals. The corresponding mean achieved dose levels in these animals were 109, 435 and 1342 mg/kg bw/day for F1 males and 160, 630 and 2262 mg/kg bw/day for F1 females, respectively. Males of the F1 generation received the diets a minimum of 10 weeks during maturation and subsequently throughout mating, gestation and until completion of the F1 female lactation phases. Females of the F1 generation were treated a minimum of 10 weeks during maturation and subsequently throughout mating, gestation and lactation phases. Between weaning and the formal start of the F1 generation, the animals continued to receive their appropriate treated diet. Animals of the F2 generation were exposed to the diets from weaning (Day 21 of age) to termination of the study on Day 70 of age. In each generation, animals administered with the high dose initially received the test material at a concentration of 15000 ppm and this was increased to 20000 ppm and finally 25000 ppm as the study progressed. Similar constituted control groups for each generation received laboratory diet enriched with Arachis oil to ensure comparable caloric intakes.

There were no treatment-related deaths and clinical signs in parental animals. During the study period, one animal of the control, mid and high dose group each were sacrificed humanely due to non-treatment-related clinical signs.

In parental animals, no adverse effects on reproductive functions were observed, as indicated by the examination of oestrous cycles, corpora lutea count, numbers of implantation sites or litter size at birth in females as well as sperm concentration, motility or morphology and homogenisation resistant spermatid counts of epididymides and testes in males. The reproductive performance as described by mating performance, fertility indices, data on gestation and parturition and sex ratio as well as lactation data, live birth and viability indices was not altered in treated animals compared to controls. An increase in organ weights (absolute and relative) of the left testes in parental males and thyroid weights in parental females was observed. The effects were considered to be incidental and not treatment-related, since they were not dose-related and not accompanied by respective changes in histopathology. No treatment-related effects were observed in parental animals at gross pathology and histopathological examination.

In animals of the F1 and F2 generation, no effects on viability and no clinical signs were observed either as consequence of maternal treatment after birth or due to of direct treatment after weaning. Body weights and food consumption in both generations of offspring were not adversely affected by treatment with the test substance. There were no obvious effects of treatment on the sexual maturation of either sex for the F1 animals receiving the test substance. Sex ratio at birth and subsequently at Day 21 of age was similar to concurrent control in all treatment groups over both generations. The slight variations in the mean ano-genital distance on Day 1 in F1 males at 1500 ppm and F2 females at 6000 ppm as well as the marginally higher visible nipple counts in F1 females at 25000 ppm, which were observed, were considered to be incidental and not treatment-related. No adverse effects were observed on reproductive performance of animals of the F1 generation.

In both generations, organ weights were not adversely altered by exposure to the test substance. Non dose-related variations in absolute and relative organ weights were observed in spleen and uterus of females of both generations.

No effects were observed on gross pathology in offspring and there were no histopathological findings observed for male and female behavioural offspring.

The neurotoxicological examination of the F0-F1 offspring of treated animals did not reveal any treatment-related effects when compared to the current F0-F1 offspring of the untreated control animals.

Based on the results of the study, the NOAEL for reproduction toxicity in parental animals is ≥ 1159 and ≥ 2200 mg/kg bw/day for males and females, respectively. For the F1 generation, the NOAEL for reproduction toxicity in males and females is ≥ 1342 and ≥ 2262 mg/kg bw/day, respectively. These doses corresponded to a concentration of 25000 ppm of the test substance in the diet.

Conclusion for reproduction toxicity

A read-across approach was applied to assess the reproductive toxicity potential of the target substance glycerides, C16-18 and C18-unsaturated mono-and di-citrates (CAS 91052-16-3). The studies on the structural analogous substances, glycerides, C8-18 and C18-unsatd, mono- and di-, acetates (CAS 91052-13-0) and Glycerides, castor-oil, mono, hydrogenated, acetates (CAS 736150-63-3) did not show treatment-related effects up to the highest tested dose level. Therefore, as the available data did not identify any hazard for reproduction toxicity, glycerides, C16-18 and C18-unsaturated mono-and di-citrates (CAS 91052-16-3) is considered to have no toxic effects to reproduction.


Justification for selection of Effect on fertility via oral route:
Hazard assessment is conducted by means of read-across from structural analogues. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between the source and target substances and overall quality assessment (refer to the endpoint discussion for further details).

Effects on developmental toxicity

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Jan 2009 - 21 Aug 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance glycerides, castor-oil, mono, hydrogenated, acetates (CAS 736150-63-3). According to the ECHA guidance document ‘Practical guide 6: How to report read-across and categories' (ECHA, 2012), the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Qualifier:
according to
Guideline:
other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 426 (Developmental Neurotoxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF, Testing Guidelines for Toxicity Studies (2-1-17), 12 Nohsan No 8147, 2000-11-24
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, UK
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley Crl:CD® (SD) IGS BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, UK
- Age at study initiation: (P) 7-8 weeks
- Weight at study initiation: (P) 216-361 g (males) and 149-249 g (females)
- Housing: All P animals were housed in groups of up to 4 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper. Mated females were housed individually (or with their litter), in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes (Datesand Ltd., Cheshire, UK). For the offspring selected to form the F1 generation, this housing procedure was repeated. The P and F1 offspring selected for assessment of developmental neurotoxicity were housed in groups of up to 4 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding.
- Diet: ground diet (Rodent PMI 5002 Diet, BCM IPS Limited, London, UK), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Each concentration of the test material was prepared by weighing an appropriate amount and mixing it with a small amount of the required volume of diet. Once this was adequately mixed the formulation was then transferred to a Hobart H800 mixer and mixed with the remaining required volume of diet.

DIET PREPARATION
- Mixing appropriate amounts with: laboratory diet
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test material in the dietary admixtures was determined by gas chromatography using an external standard technique. The dietary admixtures were sampled and analysed within 2 weeks of preparation. Homogeneity and stability determinations were performed under Harlan Laboratories Ltd. project number 2384/0006. The results showed that achieved concentrations were between 95 and 121% of nominal concentration.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating.
- After 14 days of unsuccessful pairing replacement of first male by another male or extension of the mating phase to a third week.
- After successful mating each pregnant female was caged: individually
Duration of treatment / exposure:
(P) Males: 10 weeks during maturation and throughout mating, gestation and until completion of the P female lactation phase.
(P) Females: 10 weeks during maturation and throughout mating, gestation and until Day 21 lactation phases.
(F1) Females: minimum of 10 weeks during maturation and subsequently throughout mating, gestation and lactation phases. Between weaning and the formal start of the F1 generation, the animals continued to receive their appropriate treated diet.
(F1) Males: minimum of 10 weeks during maturation and subsequently throughout mating, gestation and until completion of the F1 female lactation phases. Between weaning and the formal start of the F1 generation, the animals continued to receive their appropriate treated diet.
(F2) Males/ Females: from weaning (Day 21 of age) to termination at Day 70 of age.
Frequency of treatment:
daily, 7 days/week
Duration of test:
Day 21 of age (unselected offspring)/ Day 70 of age (selected offspring for developmental neurotoxicity)
No. of animals per sex per dose:
28 P males, 28 P females
24 F1 males, 24 F1 females
Control animals:
other: yes, laboratory diet treated with Arachis oil to ensure comparable calorific intake
Details on study design:
- Dose selection rationale: The dietary levels were based on known toxicology data including an earlier preliminary study in the rat (Project number 2384/0006)
- Other: In each generation, animals receiving the high dose level initially received the test material at a concentration of 15000 ppm and this was increased to 20000 ppm and finally 25000 ppm as the study progressed. Animals were receiving the highest inclusion level by the end of the maturation/pre-pairing phase in both generations and therefore were receiving the higher dietary inclusion by the time of the main reproductive assessment.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the week and once daily on weekends and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1, prior to treatment, weekly throughout the maturation phase of each generation and continuing for males until termination. Following pairing P/F1 females were weighted daily until mating was evident. Mated females were weighted on Day 0, 7, 14 and 21 post coitum and for females that littered on Day 1, 4, 7, 14 and 21 post partum. P/F1 offspring (selected for post-weaning assessment of developmental neurotoxicity) were weighted on Day 28, 35, 42, 49, 56, 63 and 70 of age. Body weight was recorded for all animals on the Day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption was assessed for each cage of adults during the maturation period and for males after mating/pairing. Females showing evidence of mating, food consumption was recorded for the periods Day 0-7, 7-14 and 14-21 post coitum. For females that littered, food consumption was recorded for the period covering Day 1-4, 4-7, 7-14 and 14-21 post partum.

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: daily by visual inspection for any overt change

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: cervical, thoracic, and abdominal viscera. Adrenals, coagulating gland, right epididymis, ovaries, right testis, pituitary, prostate, seminal vesicles, uterus with oviducts and cervix, vagina, gross lesions were weighed and/or preserved for histopathological examination.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
- External examinations: Yes: all unselected offspring
- Gross examination of dead pups: Yes, dead offspring was subjected to a necropsy exmination (internal and external)
Statistics:
Data were initially assessed for homogeneity of variance using Levene´s test. Where variances were shown to be homogenous, a parametric assessment of the data was performed using one way analysis of variance (ANOVA), which if significant was followed by pairwise comparisons using Dunnett´s test. Where Levene´s test showed unequal variances, the data were analysed using non-parametric methodology: Kruskal-Wallis ANOVA which, if significant, was followed by Mann-Whitney U-test. Probability values (p) are presented as follows:
p ≤ 0.001***; p ≤ 0.01**, p ≤ 0.05*, p > 0.05 (not significant).
Indices:
- Sex Ratio (% males): (Number of male offspring/ total number of offspring) x 100

- Implantation losses (%):
% pre-implantation loss = [(Number of corpora lutea - number of implantation sites) / number of corpora lutea] x 100
% post-implantation loss = [(Group number of implantation sites - number of offspring) / number of implantation sites] x 100
offspring) x 100

Live Birth and Viability Indices
- Live Birth Index (%) = (Number of offspring alive on Day 1/ number of offspring born) x 100
- Viability Index 1 (%) = (Number of offspring alive on Day 4/ number of offspring alive on Day 1) x 100
- Viability Index 2 (%) = (Number of offspring alive on Day 7/ number of offspring alive on Day 4) x 100
- Viability Index 3 (%) = (Number of offspring alive on Day 14/ number of offspring alive on Day 7) x 100
- Viability Index 4 (%) = (Number of offspring alive on Day 21/ number of offspring alive on Day 14) x 100
- Viability Index 5 (%) = (Number of offspring alive on Day 21/ number of offspring alive on Day 1) x 100

For further reproductive indices refert to 7.8.1 Toxicity to reproduction key, DuPont, 2011, rat, RL1.
Historical control data:
Historical control data for absolute and relative organ weight values, reproductive indices, fertility parameters and clinical observations were provided from Charles River (UK) Limited (2005-2008) and included in the study report.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
There were no treatment related deaths amongst maternal animals.

BODY WEIGHT AND FOOD CONSUMPTION
No adverse effects of treatment on mean bodyweight or bodyweight change was apparent in either generation for females. A significant increase in bodyweight gain during Days 1-21 in the low- and high-dose female P group was considered to be not treatment related.
No adverse effect of treatment on mean food consumption was apparent females. Food consumption of P females in the high-dose group was significantly lower during the last week of lactation. In mid-dose P females a higher food intake was observed during the last week of gestation and lactation.

ORGAN WEIGHTS
For P females of the high-dose group, absolute and bodyweight-relative thyroid weights were significantly higher. There were no significant differences in thyroid weights for F1 females or for males in either generation at this inclusion level. Given the earlier and potentially higher exposure of the F1 females to the test substance (due to low bodyweight at this stage) and the absence of any accompanying effect on thyroid weights for these animals, this finding was therefore considered to be incidental and of no toxicological significance. For P females receiving the mid and high-dose, absolute uterine weight was significantly lower; values when adjusted for the bodyweight were not significantly different from control and this finding was therefore considered to be coincidental and to reflect normal biological variation. In the low-dose P female group bodyweight-relative brain weight was significantly lower than control. In the absence of any dosage relationship, and any effect on absolute brain weight, this was considered to be incidental and unrelated to treatment.

GROSS PATHOLOGY
The majority of macroscopic necropsy findings of P females were limited to decedent animals; these deaths were considered to be unrelated to treatment.

HISTOPATHOLOGY
There were no histopathological findings observed for adults that were considered to indicate an adverse effect of the test substance at a dietary inclusion level of 25000 ppm.

POSITIVE CONTROL
Treatment at 10000 ppm DEPH was associated with clear signs of adult toxicity including notable effects on bodyweight and some organ weights. Reproduction of P animals was unaffected by treatment but shorter male ano-genital distance, increased male nipple counts and delays in attainment of sexual maturation suggested a possible endocrine disruption effect.

For further details refer to 7.8.1 Key, Danisco, 2009, rat, RL1
Dose descriptor:
NOAEL
Effect level:
>= 25 000 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 2 200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
Survival of the offspring to weaning was unaffected at the dietary concentrations by either maternal treatment or by more direct treatment when the offspring consumed the diet as they approached weaning in both generations.

CLINICAL SIGNS (OFFSPRING)
No adverse effects were observed for the test substance treated animals.

BODY WEIGHT (OFFSPRING)
There was no adverse effect of treatment on litter weights or bodyweight changes. For the F1 offspring at the high-dose litter weights were slightly lower between Days 14-21 (see Table 2 under “Any other information on results incl. tables”). For F2 offspring at the low and high-dose group bodyweight gain was slightly lower than control for both sexes between Day 7-14. These differences may reflect normal biological variation and were not considered to indicate an adverse effect of treatment.

SEXUAL MATURATION (OFFSPRING)
There were no obvious effects of treatment on the sexual maturation of either sex for the F1 animals receiving the test substance. Sex ratio at birth and subsequently at Day 21 of age was similar to concurrent control in all treatment groups. There was no adverse effect of treatment on mean ano-genital distance on Day 1 of age for either sex. For F1 males at the low-dose and F2 females at the mid-dose, mean ano-genital distance on Day 1 of age was slightly shorter (see Table 3 under “Any other information on results incl. tables”). These decreases were considered to reflect normal biological variation and were considered to be unrelated to treatment. There was no adverse effect of treatment on visible nipple counts on Days 11-15 of age for either sex in either generation. Only for F1 females at the high-dose visible nipple counts were marginally higher than control only at Day 11 of age, therefore this effect was considered to be incidental and unrelated to treatment (see Table 4 under "Any other information on results incl. tables").

GROSS PATHOLOGY (OFFSPRING)
The macroscopic abnormalities observed for both decedent and terminal kill offspring were typical for the age examined and neither the incidence or distribution of these findings indicated any adverse effect of treatment with the test substance in either generation.

POSITIVE CONTROL (OFFSPRING)
Adverse effects on offspring survival and growth were apparent and offspring failed to thrive following separation from the parent female at weaning. Effects on brain, spleen, thymus and, for females, uterus weight were apparent for the F1 offspring. Necropsy revealed enlarged liver and kidneys, small testes, epididymides and seminal vesicles for male F1 animals.
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
>= 25 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity: overall effects
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
>= 1 342 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
>= 2 262 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1. Overall achieved intakes during each generation for test material treated groups.

Dietary Concentration

Males (mg/kg bw/day)
   F0   F1   

Females (mg/kg bw/day)
Maturation Gestation Lactation


 F0     F1     F0      F1    F0    F1

1500 ppm 82 109 106 135 101 108 231 238
6000 ppm 324 435 431 540 411 434 919 918
25000 ppm 1159 1342 1392 1493 1665 1697 3544 3596

Table 2. Table for reproductive performance.

Parameter

Control group

Low-dose group

1500 ppm

Mid-dose group 6000 ppm

High-dose group 25000 ppm

Live Birth Index

F0-F1

F1-F2

 

99.3

99.6

 

98.2

98.3

 

98.4

99.5

 

98.4

99.5

Pre-Implantation Loss [%]

F0-F1

F1-F2

 

5.4 ± 4.6

5.5 ± 8.7

 

6.8 ± 7.4

4.9 ± 8.8

 

5.7 ± 4.0

5.4 ± 9.8

 

5.4 ± 6.1

1.7 ± 3.1

Post-Implantation Loss [%]

F0-F1

F1-F2

 

3.1 ± 6.5

7.0 ± 8.7

 

3.7 ± 5.5

4.2 ± 5.2

5.0 ± 7.4

4.9 ± 7.8

10.8 ± 16.2**

7.7 ± 13.8

Total Litter Weight (g)

F1 Day 1

F1 Day 14

F1 Day 21

F2 Day 1

F2 Day 14

F2 Day 21

 

95.1

389.5

600.0

87.3

363.2

564.2

 

89.6

362.6

567.4

101.0

384.1

599.3

 

92.4

368.8

584.8

95.6

368.9

570.4

 

88.9

343.7**

523.8**

98.0

360.0

555.2

Total number of Corpora Lutea

F0-F1

F1-F2

 

15.7 ± 1.6

15.3 ± 2.3

 

15.7 ± 2.7

17.2 ± 2.2

 

15.7 ± 1.8

16.5 ± 2.9

 

15.9 ± 2.0

16.3 ± 3.8

Total number of Implantation Sites

F0-F1

F1-F2

 

 

14.8 ± 1.7

14.4 ± 2.5

 

 

14.5 ± 2.7

16.3 ± 1.8

  

14.8 ± 2.0

15.4 ± 2.4

 

15.0 ± 1.7

16.0 ± 3.7

Total Number of Offspring Born

F0-F1

F1-F2

 

14.4 ± 2.1

13.5 ± 2.9

  

14.0 ± 2.6

15.5 ± 1.7

14.2 ± 2.4

14.6 ± 2.5

  

13.4 ± 1.7

14.8 ± 4.0

Sex ratio (% male at birth)

51.9

45.3

46.8

51.8

Table 3. Ano-Genital distance of offspring - group mean litter values.

Ano-genital distance (mm) on Day 1 post partum

Control group

Low-dose group

1500 ppm

Mid-dose group 6000 ppm

High-dose group 25000 ppm

Males

F1

F2

3.12 ± 0.34

3.01 ± 0.33

2.95* ± 0.40

3.03 ± 0.33

3.12 ± 0.46

3.01 ± 0.33

3.13 ± 0.36

3.11 ± 0.31

Females

F1

F2

1.60 ± 0.34

1.47 ± 0.19

1.65 ± 0.44

1.51± 0.24

 

1.49 ± 0.31

1.33± 0.14**

 

1.67 ± 0.34

1.49± 0.22

p** ≤ 0.01

Table 4. Nipple counts for the F1 generation at Day 11 of age.

Group

Mean Nipple count at Day 11 of age

Male

Female

Control

0.0 ± 0.0

9.4 ± 1.8

Low dose group

0.1 ± 0.5

10.4 ± 1.5

Mid-dose group

0.0 ± 0.0

9.9 ± 1.5

High-dose group

0.1 ± 0.3

10.6 ± 1.4*

p* ≤ 0.05

Conclusions:
The test substance had no effect on intrauterine development.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Feb - 07 April 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP-Guideline study, tested with the source substance glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS 91052-13-0). According to the ECHA guidance document ‘Practical guide 6: How to report read-across and categories' (ECHA, 2012), the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
only external abnormalities and mainly macroscopic examination of soft tissues performed, no skeletal examination of pups
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
- Age at study initiation: approximately 12 weeks.
- Weight at study initiation: 277-353 g (males), 180-228 g (females)
- Housing : 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). This was also applicable for Main females and Recovery animals throughout the complete treatment period. Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 (actual range: 19.7-21.9)
- Humidity (%): 40-70 (actual range: 22-71)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. The testing laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6h prior to dosing and were homogenized to a visually acceptable level.
Adjustment was made for the density of the test substance (0.944), and the specific gravity of the vehicle (1.125).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Purity: polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany)

Dose volume: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6h at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤10%) and formulations at the entire range were stable when stored at room temperature for at least 6h.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type).
Duration of treatment / exposure:
41-49 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.
Frequency of treatment:
daily, 7 days/week
Duration of test:
41-49 days
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 10-day dose range finding study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: Yes

POST-MORTEM EXAMINATIONS: Yes

For further details see Section 7.5.1
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: No

The numbers of former implantation sites and corpora lutea were recorded for all paired females.
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes, partly (see details below)
- Skeletal examinations: No
- Head examinations: No

Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made in all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Pups surviving to planned termination were killed by decapitation on lactation Day 5.
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue as well as pups from females that were killed in extremis were preserved in 10% buffered formalin for possible further examination.

Statistics:
See Section 7.5.1
Indices:
For each group the following calculations were performed:
- Mating (%): Number of females mated/Number of females paired x 100
- Fertility index (%): Number of pregnant females/Number of females paired x 100
- Conception index (%): Number of pregnant females/Number of females mated x 100
- Gestation index (%): Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100
Details on maternal toxic effects:
Maternal toxic effects:no effects
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Number of live pups: 432
Mortality: 1/119, 1/116. 1/126 and 1/71 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. This is not considered to be treatment-related, as this falls within the expected range for this strain and age.
A statistically significant lower mean number of living pups at first litter check observed at 1000 mg/kg/day was due to a low number of pups (5 in total) for one female. Since the number of pups of other females of this dose group was within the range considered normal, no toxicological relevance was ascribed to this change.
Viability index, body weights of pups, external examination for abnormalities and clinical signs of pups did not indicate any treatment-related abnormalities
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Viability index, body weights of pups, external examination for abnormalities and clinical signs of pups did not indicate any treatment-related abnormalities
Abnormalities:
not specified
Developmental effects observed:
not specified
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Comparable to guideline study with acceptable restrictions. Parenteral route of application, only two dose levels, number of corpora lutea not reported. Justification for grouping of substances and read-across: The study is selected as a suitable study for the assessment of potential developmental toxicity/teratogenic effects within the Glycerides category. Read-across from Medium Chain Triglycerides (MCT) (no CAS) is conducted on the basis of Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 by application of the group/category concept (i.e. the physicochemical, toxicological and ecotoxicological properties of a group of substances are likely to be similar or follow a regular pattern as a result of structural similarity). Human health effects with regard to toxicity to reproduction were predicted from data for reference substances within the category by interpolation. The similarities between the members of the Glycerides category are based on the common functional groups, the common precursors and the likelihood of common breakdown products, and a constant pattern in the changing of the potency of the properties across the category. The selected study fulfils the requirements laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 for read-across, i.e. the results are adequate for the purpose of classification and labelling and/or risk assessment; have adequate and reliable coverage of the key parameters addressed in the corresponding test method referred to in Article 13(3); cover an exposure duration comparable to or longer than the corresponding test method referred to in Article 13(3); and adequate and reliable documentation of the applied method is provided. A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Section 13).
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
parenteral route of application, only two dose levels, number of corpora lutea not reported
GLP compliance:
not specified
Limit test:
no
Species:
rabbit
Strain:
other: Hra:(NZW)SPF rabbits
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HRP, Inc., Denver, PA, USA
- Age at study initiation: 5.5 - 6.5 months
- Weight at study initiation: 3300 - 4446 g
- Housing: individually housed in suspended stainless steel cages
- Diet: certified rabbit diet 5322 (PMI Feeds. Inc.), ad libitum; except during dose administration
- Water: ad libitum; except during dose administration

ENVIRONMENTAL CONDITIONS
Environmental controls in the animal rooms were set to maintain temperature, relative humidity, and light/dark cycle (no further information)
Route of administration:
intravenous
Vehicle:
other: 20% lipid emulsion, no further specified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose was administered daily to rabbits via a marginal ear vein using an indwelling catheter [Insyte-W (24-gauge, 3/4-inch intravenous catheter and needle unit attached to a 30-inch extension set, Abbott Laboratories)]. Doses for rabbits were delivered using syringe pumps. The rabbits were acclimated to the dose restraint apparatus for 2.5 and 5 h on the 2 days preceding initiation of dosing.


Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Mated rabbits were received on gestation day 2 or 3 in two consecutive weekly shipments and were observed at arrival for abnormalities indicative of health problems.
Duration of treatment / exposure:
(P): 13 days during gestation
Frequency of treatment:
5 h/day, 7 days/week
Duration of test:
Day 29 of gestation / post mating
No. of animals per sex per dose:
15
Control animals:
other: 0.9% saline
Details on study design:
- Dose selection rationale: The dose rate was based on previous preclinical studies (unpublished). The 1000 mg/kg dose approximates the proposed clinical dosage. The 4280 mg/kg dose is the highest dose administered in preclinical studies that did not produce narcosis (unpublished).

- Selection of exposurre route: The intravenous route of administration was used because the lipid emulsion is intended for intravenous human administration as a component of parenteral nutrition.

- Dose volumes: 5 and 21.4 mL/kg

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily (AM and PM) for mortality and moribundity. In addition, rabbits were observed once daily for clinical observations. On dosing days, animals were observed predose, immediately (within 5 min) postdose, and approximately 1 h after completion of dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: daily and on GD 7 through 29

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Feed consumption data were collected daily beginning on GD 6.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all. Live fetuses were weighed, examined externally, then euthanatized.
- Soft tissue examinations: Yes: all. The internal organs of the rabbit fetuses were examined in the fresh state for variations and malformations, and the sex was determined. Following completion of fetal examinations, soft tissue malformations were preserved in 10% phosphate-buffered formalin.
- Skeletal examinations: Yes: all. Viscera were removed and discarded, and fetuses were processed and examined for skeletal variations and malformations; skeletal specimens were retained in glycerine.
- Head examinations: Yes: all. A mid-corona slice of the head was made to expose the internal structure of the brain for examination; the eyes were excised and examined.
Statistics:
The litter was the experimental unit for evaluation. All comparisons were made with the control group (Group 1). For rabbit data, Levene's test (Levene, 1960) was done to test for variance homogeneity. In the case of heterogeneity of variance at p « 0.05, rank transformation was used to stabilize the variance. Analysis of variance [ANOVA (Winer, 1971a)] was done on the homogeneous or transformed data. If the ANOVA was significant, Dunnett's t test (Dunnett, 1964) was used for pairwise comparisons between groups. One-way ANOVA was used to analyse body weights, body weight changes, feed consumption, and cesarcan section data. As appropriate, rabbit fetal abnormality data were analysed by the Cochran-Armitage test (Thalcur et al, 1985) for trend and departure and by the Fisher-Irwin exact test (Thalcur et al., 1985). One-way analysis of covariance [ANCOVA (Winer, 1971b)] was used to analyse fetal body weights (males, females, and combined) with the number of fetuses in the litter as the covariate. As appropriate, for values calculated to analyse litter data or mean fetal weight data, values were first derived within the litter, and the group mean values were derived as a mean of individual litter values. Group comparisons were analysed at the 5.0 and 1.0% two-tailed probability levels.
Indices:
Early and late resorptions, % fetuses dead/live, % postimplantation loss
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: at the highest dose group

Details on maternal toxic effects:
Survival was 100% for the 1000 mg lipid/kg group. One animal in the control group died on GD 11 and one animal given 4280 mg lipid/kg was sacrificed after aborting on GD 20. The animal that aborted had the lowest food consumption in the group. Decreased food consumption, resulting in a decline in the health of this animal, may have contributed to the abortion. The abortion was not considered to be test article-related as it was in the range of historical control incidence. There were no remarkable necropsy findings for either animal. Clinical observations in the test article-treated groups were limited to faecal findings (i.e., increased incidence of few or no faeces). Three animals each at 4280 mg lipid/kg had no faecal output for 1 day during the dosing period.
The data on maternal body weight changes as taken from the tabellary reporting showed a significant loss of body weights during gestational day 7 - 20 in maternal rabbits accompanied by reduced food consumption in the high dose group.
The body weight gain during GD 7-20 was found to be 95.4 g in control animals and 119.6 g in animals dosed with 1000 mg/kg bw/day, whereas at the same time animals dosed with 4280 mg/kg bw/day showed a significant loss of body weight of - 124.8 g (p < 0.01).
Food consumption in the highest dose group was only 50% of the control during GD 12 - 13 and only 10% of the control during GD19 - 20. Food consumption continued to be significantly lower for the highest dose group during the early post-treatment period (GD 20 to 24), but recovery was noted at a later interval (GD 24 to 29). The decreased food consumption observed in this study was an expected occurrence based on the high-caloric nature of the test article. There were no test article-related findings at necropsy. All pregnant animals had at least one viable foetus at scheduled caesarean section on GD 29 (i.e., no dams had total resorptions).

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Remarks:
(i.v. application)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: at the highest dose group

Details on embryotoxic / teratogenic effects:
The mean percentage of total resorptions/litter (postimplantation loss) was significantly higher and the mean percentage of live foetuses/litter was correspondingly lower in the 4280 mg lipid/kg group. Mean covariate-adjusted foetal body weights (males, females, and combined) for the 4280 mg lipid/kg group were significantly lower.
The proportion of foetuses and litters in the 4280 mg lipid/kg group with external morphological abnormalities was significantly higher than that of the control group.
The most notable findings were rachischisis and short tail seen in three and two high-dose litters, respectively. Single incidences of the following malformations were also present in the high-dose group: exencephaly, ablepharia, exophthalmus, and ectrodactyly. Single litter incidences of carpal flexure, tarsal flexure, and malpositioned shoulders were noted for the 1000 mg lipid/kg group; however, these were not considered to be test article-related. No external abnormalities were noted for the control group.
The total incidence of litters in the 1000 mg lipid/kg group with foetuses having soft tissue abnormalities was significantly higher than that of the control group; however, no significant differences were present when individual malformations and variations were analysed statistically. Although the incidence of litters in the 4280 mg lipid/kg group with viscerally abnormal foetuses was also higher than controls, the difference was not statistically significant. In general, soft tissue abnormalities were present in the test article-treated groups as single foetal or litter incidences restricted to three or four litters/group, suggesting a litter-related occurrence.
Because no soft tissue abnormalities (with the exception of absent azygous lobe of the lung) were noted for the control litters, the relationship of the abnormalities in the test article groups to the test article is inconclusive.
The proportions of foetuses with variations in the 4280 mg lipid/kg group [skull bones unossified, more than 26 presacral vertebrae, and 12 full pairs of ribs (litter incidence was also greater)] and total skeletal abnormalities were significantly higher than those in the control group. The proportions of litters in this group with foetuses having misaligned sternebrae, more than 12 full pairs of ribs, and fused ribs were also significantly higher than those in the control group.
Though not statistically significant, there was a notable increase in the number of high-dose foetuses/litter with malformations of the vertebral column. The anomalies were varied in location (cervical, thoracic, sacral, and caudal) and type (malformed, misaligned, absent, and fused). The incidence of any one of these findings was not markedly higher than that of the controls (usually only one litter was affected with a given abnormality); however, when combined, the incidence of foetuses/litters from the high-dose group with vertebral column malformations was notably higher than that of the control group.

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Remarks:
(i.v. application)
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Effect level:
4 280 mg/kg bw/day
Based on:
test mat.
Remarks:
(i.v. application)
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Cesarean section examinations of rabbits revealed higher postimplantation loss (i.e., increased incidence of resorptions) and, correspondingly, fewer live foetuses at the 4280 mg lipid/kg level. Mean foetal weights were lower for this group as well, and more litters in the group tended to have foetuses with morphological anomalies than were seen in the control group. Reduced foetal weights may have been secondary to the decreased maternal food consumption observed at this dose level.

Because treated females were consuming significantly less food than control females, the foetal effects noted for rabbits (especially the postimplantation loss and decreased foetal weights) may have been due to dietary deprivation, as opposed to a direct effect by the test article. Increased incidence of abortion and implant resorption among pregnant rabbits subjected to dietary deprivation have been reported (Matsazawa et at, 1980). There were no related patterns of major malformations for foetuses. Skeletal variations were present in all litters, including control and treated groups. Diverse abnormalities were seen that may have been associated with maternal toxicity (Khera, 1984; Manson, 1986).

Administration of the test article to rabbits at 1000 or 4280 mg lipid/kg resulted in a NOEL for developmental toxicity greater than or equal to 1000 mg lipid/kg but less than 4280 mg lipid/kg based on the adverse foetal findings (increased postimplantation loss, lower foetal body weights, and higher incidence of morphological anomalies) seen at the 4280 mg lipid/kg level. Administration of the test article at the highest dose also resulted in lower maternal food consumption and loss of body weight, therefore the observed foetal effects were probably the result of dietary deprivation, maternal toxicity or both, rather than a direct teratogenic effect of the test article.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Comparable to guideline study with acceptable restrictions. Parenteral route of application, only two dose levels, number of corpora lutea not reported. Justification for grouping of substances and read-across: The study is selected as a suitable study for the assessment of potential developmental toxicity/teratogenic effects within the Glycerides category. Read-across from Medium Chain Triglycerides (MCT) (no CAS) is conducted on the basis of Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 by application of the group/category concept (i.e. the physicochemical, toxicological and ecotoxicological properties of a group of substances are likely to be similar or follow a regular pattern as a result of structural similarity). Human health effects with regard to toxicity to reproduction were predicted from data for reference substances within the category by interpolation. The similarities between the members of the Glycerides category are based on the common functional groups, the common precursors and the likelihood of common breakdown products, and a constant pattern in the changing of the potency of the properties across the category. The selected study fulfils the requirements laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 for read-across, i.e. the results are adequate for the purpose of classification and labelling and/or risk assessment; have adequate and reliable coverage of the key parameters addressed in the corresponding test method referred to in Article 13(3); cover an exposure duration comparable to or longer than the corresponding test method referred to in Article 13(3); and adequate and reliable documentation of the applied method is provided. A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Section 13).
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
parenteral route of application, only two dose levels, number of corpora lutea not reported
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: Crl:CD BR rats
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI, USA
- Weight at study initiation: 150 - 200 g
- Housing: individually housed in suspended stainless steel cages
- Diet: Certified Rodent Chow 5002 meal (PMI Feeds, Inc.), ad libitum; except during dose administration
- Water: ad libitum; except during dose administration

ENVIRONMENTAL CONDITIONS
Environmental controls in the animal rooms were set to maintain temperature, relative humidity, and light/dark cycle
Route of administration:
intravenous
Vehicle:
other: 20% lipid emulsion was created, no further specified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose was administered daily to rats by intravenous infusion via a caudal vein using a Quik-Cath Teflon catheter connected to a syringe using an extension set. Doses for rats and rabbits were delivered using syringe pumps.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Time-mated Crl:CD BR rats were shipped by the supplier on GD 4.
Duration of treatment / exposure:
Day 6-15 of gestation
Frequency of treatment:
4 h/day, 7 days/week
Duration of test:
Day 20 of gestation / post mating
No. of animals per sex per dose:
25 or 29 females (P)
Control animals:
other: 0.9% saline
Details on study design:
- Dose selection rationale: The dose rate was based on previous preclinical studies (unpublished). The 1 g/kg dose approximates the proposed clinical dosage. The 4.28 g/kg dose is the highest dose administered in preclinical studies that did not produce narcosis (unpublished).
- Selection of exposure route: The intravenous route of administration was used because the lipid emulsion is intended for intravenous human administration as a component of parenteral nutrition. The dose was administered daily to rats by intravenous infusion for approximately 4 h/day on GD 6 through 15 via a caudal vein using a Quik-Cath Teflon catheter connected to a syringe using an extension set.
- Dose volumes: 5 and 21.4 mL/kg bw
Maternal examinations:
CLINICAL SIGNS AND CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily (AM and PM) for mortality and moribundity. On dosing days, animals were observed predose, immediately (within 5 min) postdose, and approximately 1 h after completion of dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: daily on GD 5 - 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Food consumption data were collected daily beginning on the day of receipt

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day GD 20


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: half litter; live fetuses were weighed, examined externally, sexed, then euthanatised
- Soft tissue examinations: half litter; Following completion of fetal examinations, soft tissue malformations were preserved in 10% phosphate-buffered formalin
- Skeletal examinations: half litter; Viscera were removed and discarded, and fetuses were processed and examined for skeletal variations and malformations; skeletal specimens were retained in glycerine
- Head examinations: half litter; A mid-coronal slice of the head was made to expose the internal structure of the brain for examination; the eyes were excised and examined.
Statistics:
The litter was the experimental unit for evaluation. All comparisons were made with the control group (Group 1). For rats, feed consumption, dam body weights, and fetal body weights were summarized with means and standard deviations calculated using an Excel spreadsheet program (Microsoft Corporation). For rabbit data, Levene's test (Levene, 1960) was done to test for variance homogeneity. In the case of heterogeneity of variance at p « 0.05, rank transformation was used to stabilize the variance. Analysis of variance [ANOVA (Winer, 1971a)] was done on the homogeneous or transformed data. If the ANOVA was significant, Dunnett's t test (Dunnett, 1964) was used for pairwise comparisons between groups. One-way ANOVA was used to analyze body weights, body weight changes, feed consumption, and cesarcan section data. As appropriate, rat and rabbit fetal abnormality data were analyzed by the Cochran-Armitage test (Thalcur et al, 1985) for trend and departure and by the Fisher-Irwin exact test (Thalcur et al., 1985). One-way analysis of covariance [ANCOVA (Winer, 1971b)] was used to analyze fetal body weights (males, females, and combined) with the number of fetuses in the litter as the covariate. As appropriate, for values calculated to analyze litter data or mean fetal weight data, values were first derived within the litter, and the group mean values were derived as a mean of individual litter values. Group comparisons were analyzed at the 5.0 and 1.0% two-tailed probability levels.
Indices:
early and late resorptions, % fetuses dead/live, % postimplantation loss
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: effects in the highest dose group

Details on maternal toxic effects:
The only test article-related findings observed were associated with tail lesions in both test article groups and the occasional occurrence of red tinged urine (8 of 29) and vaginal bleeding (1 of 29) in the high-dose group. The tail findings were predominantly those of discoloration and ulceration. Incidences were 1/25, 14/25, and 23/29 for these tail lesions in the control, low-, and high-dose groups, respectively, and ranged from mild to severe with some necrosis and partial loss of the tail.
These findings were generally considered to be related to occurrences of observed extravasation of the MCT:LCT lipid test article into perivascular areas. Evaluation of urine collected from one high-dose animal suggested a bacterial infection of the urogenital tract. Clinical observations for this animal included occasional red-tinged urine and vaginal bleeding. This rat was noted as having a large urinary bladder stone and kidney hydronephrosis at necropsy.
There were no marked differences in mean body weights or feed consumption for the low-dose group compared with those of the control group. However, the high-dose group consistently exhibited lower body weights beginning 1 day after dose administration throughout the remainder of the study. Feed consumption was also notably lower for nine of the ten days during dosing, with an increase in feed consumption after completion of dose administration (GD 15). The decrease in feed consumption at the high-dose level was expected based on the high-caloric nature of the test article.
Necropsy findings were primarily related to tail effects and were observed for most rats in the high-dose group and some rats in the low-dose group. In addition to tail effects, there was a trend toward an increasing incidence of necropsy findings in the high-dose group, including enlarged lymph nodes, enlarged spleen, hydronephrosis/enlarged renal pelvis, small thymus, and small red lung foci. These changes indicated that the high-dose group was likely exhibiting test article effects.
There was a slight trend toward decreasing mean gravid uterine weights in proportion to increasing test article dose; however, due to the large variability between groups, group mean uterine weights appeared to be similar. With the exception of one control dam, all females were pregnant and had at least one viable fetus/litter (i.e., no dams had total resorptions).
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Remarks:
(i.v. application)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no significant group differences in preimplantation or postimplantation loss or in the mean percentage of live or resorbed fetuses; no dead fetuses were present. Mean fetal sex ratios of the test article-treated groups were comparable with those of controls. There were no apparent effects on mean fetal body weight (combined, males, or females). There were no test article-related fetal external, soft tissue, or skeletal observations.
A high incidence of folded retina in control and test article-treated groups was attributed to shrinkage of the retina during storage in alcohol prior to being transferred to Bouin's fixative.
Omphalocele and cleft palate were observed in one fetus each in the control and high-dose groups, respectively. The only fetal skeletal malformation observed (malformed/misshapen skull bones) was present in one fetus each from two control litters. Fetal skeletal variations were present in control and test article-treated groups in a nondose-related pattern.

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
4 280 mg/kg bw/day
Based on:
test mat.
Remarks:
(i.v. application)
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Upon intravenous application of 4280 mg/kg bw/day during gestational Days 6 - 15, medium chain triglycerides exerted no teratogenic effects in rats.
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Reason / purpose:
read-across source
Species:
rat
Strain:
other: Sprague-Dawley Crl:CD® (SD) IGS BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, UK
- Age at study initiation: (P) 7-8 weeks
- Weight at study initiation: (P) 216-361 g (males) and 149-249 g (females)
- Housing: All P animals were housed in groups of up to 4 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper. Mated females were housed individually (or with their litter), in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes (Datesand Ltd., Cheshire, UK). For the offspring selected to form the F1 generation, this housing procedure was repeated. The P and F1 offspring selected for assessment of developmental neurotoxicity were housed in groups of up to 4 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding.
- Diet: ground diet (Rodent PMI 5002 Diet, BCM IPS Limited, London, UK), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Each concentration of the test material was prepared by weighing an appropriate amount and mixing it with a small amount of the required volume of diet. Once this was adequately mixed the formulation was then transferred to a Hobart H800 mixer and mixed with the remaining required volume of diet.

DIET PREPARATION
- Mixing appropriate amounts with: laboratory diet
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test material in the dietary admixtures was determined by gas chromatography using an external standard technique. The dietary admixtures were sampled and analysed within 2 weeks of preparation. Homogeneity and stability determinations were performed under Harlan Laboratories Ltd. project number 2384/0006. The results showed that achieved concentrations were between 95 and 121% of nominal concentration.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating.
- After 14 days of unsuccessful pairing replacement of first male by another male or extension of the mating phase to a third week.
- After successful mating each pregnant female was caged: individually
Duration of treatment / exposure:
(P) Males: 10 weeks during maturation and throughout mating, gestation and until completion of the P female lactation phase.
(P) Females: 10 weeks during maturation and throughout mating, gestation and until Day 21 lactation phases.
(F1) Females: minimum of 10 weeks during maturation and subsequently throughout mating, gestation and lactation phases. Between weaning and the formal start of the F1 generation, the animals continued to receive their appropriate treated diet.
(F1) Males: minimum of 10 weeks during maturation and subsequently throughout mating, gestation and until completion of the F1 female lactation phases. Between weaning and the formal start of the F1 generation, the animals continued to receive their appropriate treated diet.
(F2) Males/ Females: from weaning (Day 21 of age) to termination at Day 70 of age.
Frequency of treatment:
daily, 7 days/week
Duration of test:
Day 21 of age (unselected offspring)/ Day 70 of age (selected offspring for developmental neurotoxicity)
No. of animals per sex per dose:
28 P males, 28 P females
24 F1 males, 24 F1 females
Control animals:
other: yes, laboratory diet treated with Arachis oil to ensure comparable calorific intake
Details on study design:
- Dose selection rationale: The dietary levels were based on known toxicology data including an earlier preliminary study in the rat (Project number 2384/0006)
- Other: In each generation, animals receiving the high dose level initially received the test material at a concentration of 15000 ppm and this was increased to 20000 ppm and finally 25000 ppm as the study progressed. Animals were receiving the highest inclusion level by the end of the maturation/pre-pairing phase in both generations and therefore were receiving the higher dietary inclusion by the time of the main reproductive assessment.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the week and once daily on weekends and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1, prior to treatment, weekly throughout the maturation phase of each generation and continuing for males until termination. Following pairing P/F1 females were weighted daily until mating was evident. Mated females were weighted on Day 0, 7, 14 and 21 post coitum and for females that littered on Day 1, 4, 7, 14 and 21 post partum. P/F1 offspring (selected for post-weaning assessment of developmental neurotoxicity) were weighted on Day 28, 35, 42, 49, 56, 63 and 70 of age. Body weight was recorded for all animals on the Day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption was assessed for each cage of adults during the maturation period and for males after mating/pairing. Females showing evidence of mating, food consumption was recorded for the periods Day 0-7, 7-14 and 14-21 post coitum. For females that littered, food consumption was recorded for the period covering Day 1-4, 4-7, 7-14 and 14-21 post partum.

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: daily by visual inspection for any overt change

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: cervical, thoracic, and abdominal viscera. Adrenals, coagulating gland, right epididymis, ovaries, right testis, pituitary, prostate, seminal vesicles, uterus with oviducts and cervix, vagina, gross lesions were weighed and/or preserved for histopathological examination.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
- External examinations: Yes: all unselected offspring
- Gross examination of dead pups: Yes, dead offspring was subjected to a necropsy exmination (internal and external)
Statistics:
Data were initially assessed for homogeneity of variance using Levene´s test. Where variances were shown to be homogenous, a parametric assessment of the data was performed using one way analysis of variance (ANOVA), which if significant was followed by pairwise comparisons using Dunnett´s test. Where Levene´s test showed unequal variances, the data were analysed using non-parametric methodology: Kruskal-Wallis ANOVA which, if significant, was followed by Mann-Whitney U-test. Probability values (p) are presented as follows:
p ≤ 0.001***; p ≤ 0.01**, p ≤ 0.05*, p > 0.05 (not significant).
Indices:
- Sex Ratio (% males): (Number of male offspring/ total number of offspring) x 100

- Implantation losses (%):
% pre-implantation loss = [(Number of corpora lutea - number of implantation sites) / number of corpora lutea] x 100
% post-implantation loss = [(Group number of implantation sites - number of offspring) / number of implantation sites] x 100
offspring) x 100

Live Birth and Viability Indices
- Live Birth Index (%) = (Number of offspring alive on Day 1/ number of offspring born) x 100
- Viability Index 1 (%) = (Number of offspring alive on Day 4/ number of offspring alive on Day 1) x 100
- Viability Index 2 (%) = (Number of offspring alive on Day 7/ number of offspring alive on Day 4) x 100
- Viability Index 3 (%) = (Number of offspring alive on Day 14/ number of offspring alive on Day 7) x 100
- Viability Index 4 (%) = (Number of offspring alive on Day 21/ number of offspring alive on Day 14) x 100
- Viability Index 5 (%) = (Number of offspring alive on Day 21/ number of offspring alive on Day 1) x 100

For further reproductive indices refert to 7.8.1 Toxicity to reproduction key, DuPont, 2011, rat, RL1.
Historical control data:
Historical control data for absolute and relative organ weight values, reproductive indices, fertility parameters and clinical observations were provided from Charles River (UK) Limited (2005-2008) and included in the study report.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
There were no treatment related deaths amongst maternal animals.

BODY WEIGHT AND FOOD CONSUMPTION
No adverse effects of treatment on mean bodyweight or bodyweight change was apparent in either generation for females. A significant increase in bodyweight gain during Days 1-21 in the low- and high-dose female P group was considered to be not treatment related.
No adverse effect of treatment on mean food consumption was apparent females. Food consumption of P females in the high-dose group was significantly lower during the last week of lactation. In mid-dose P females a higher food intake was observed during the last week of gestation and lactation.

ORGAN WEIGHTS
For P females of the high-dose group, absolute and bodyweight-relative thyroid weights were significantly higher. There were no significant differences in thyroid weights for F1 females or for males in either generation at this inclusion level. Given the earlier and potentially higher exposure of the F1 females to the test substance (due to low bodyweight at this stage) and the absence of any accompanying effect on thyroid weights for these animals, this finding was therefore considered to be incidental and of no toxicological significance. For P females receiving the mid and high-dose, absolute uterine weight was significantly lower; values when adjusted for the bodyweight were not significantly different from control and this finding was therefore considered to be coincidental and to reflect normal biological variation. In the low-dose P female group bodyweight-relative brain weight was significantly lower than control. In the absence of any dosage relationship, and any effect on absolute brain weight, this was considered to be incidental and unrelated to treatment.

GROSS PATHOLOGY
The majority of macroscopic necropsy findings of P females were limited to decedent animals; these deaths were considered to be unrelated to treatment.

HISTOPATHOLOGY
There were no histopathological findings observed for adults that were considered to indicate an adverse effect of the test substance at a dietary inclusion level of 25000 ppm.

POSITIVE CONTROL
Treatment at 10000 ppm DEPH was associated with clear signs of adult toxicity including notable effects on bodyweight and some organ weights. Reproduction of P animals was unaffected by treatment but shorter male ano-genital distance, increased male nipple counts and delays in attainment of sexual maturation suggested a possible endocrine disruption effect.

For further details refer to 7.8.1 Key, Danisco, 2009, rat, RL1
Dose descriptor:
NOAEL
Effect level:
>= 25 000 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 2 200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
Survival of the offspring to weaning was unaffected at the dietary concentrations by either maternal treatment or by more direct treatment when the offspring consumed the diet as they approached weaning in both generations.

CLINICAL SIGNS (OFFSPRING)
No adverse effects were observed for the test substance treated animals.

BODY WEIGHT (OFFSPRING)
There was no adverse effect of treatment on litter weights or bodyweight changes. For the F1 offspring at the high-dose litter weights were slightly lower between Days 14-21 (see Table 2 under “Any other information on results incl. tables”). For F2 offspring at the low and high-dose group bodyweight gain was slightly lower than control for both sexes between Day 7-14. These differences may reflect normal biological variation and were not considered to indicate an adverse effect of treatment.

SEXUAL MATURATION (OFFSPRING)
There were no obvious effects of treatment on the sexual maturation of either sex for the F1 animals receiving the test substance. Sex ratio at birth and subsequently at Day 21 of age was similar to concurrent control in all treatment groups. There was no adverse effect of treatment on mean ano-genital distance on Day 1 of age for either sex. For F1 males at the low-dose and F2 females at the mid-dose, mean ano-genital distance on Day 1 of age was slightly shorter (see Table 3 under “Any other information on results incl. tables”). These decreases were considered to reflect normal biological variation and were considered to be unrelated to treatment. There was no adverse effect of treatment on visible nipple counts on Days 11-15 of age for either sex in either generation. Only for F1 females at the high-dose visible nipple counts were marginally higher than control only at Day 11 of age, therefore this effect was considered to be incidental and unrelated to treatment (see Table 4 under "Any other information on results incl. tables").

GROSS PATHOLOGY (OFFSPRING)
The macroscopic abnormalities observed for both decedent and terminal kill offspring were typical for the age examined and neither the incidence or distribution of these findings indicated any adverse effect of treatment with the test substance in either generation.

POSITIVE CONTROL (OFFSPRING)
Adverse effects on offspring survival and growth were apparent and offspring failed to thrive following separation from the parent female at weaning. Effects on brain, spleen, thymus and, for females, uterus weight were apparent for the F1 offspring. Necropsy revealed enlarged liver and kidneys, small testes, epididymides and seminal vesicles for male F1 animals.
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
>= 25 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity: overall effects
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
>= 1 342 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
>= 2 262 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1. Overall achieved intakes during each generation for test material treated groups.

Dietary Concentration

Males (mg/kg bw/day)
   F0   F1   

Females (mg/kg bw/day)
Maturation Gestation Lactation


 F0     F1     F0      F1    F0    F1

1500 ppm 82 109 106 135 101 108 231 238
6000 ppm 324 435 431 540 411 434 919 918
25000 ppm 1159 1342 1392 1493 1665 1697 3544 3596

Table 2. Table for reproductive performance.

Parameter

Control group

Low-dose group

1500 ppm

Mid-dose group 6000 ppm

High-dose group 25000 ppm

Live Birth Index

F0-F1

F1-F2

 

99.3

99.6

 

98.2

98.3

 

98.4

99.5

 

98.4

99.5

Pre-Implantation Loss [%]

F0-F1

F1-F2

 

5.4 ± 4.6

5.5 ± 8.7

 

6.8 ± 7.4

4.9 ± 8.8

 

5.7 ± 4.0

5.4 ± 9.8

 

5.4 ± 6.1

1.7 ± 3.1

Post-Implantation Loss [%]

F0-F1

F1-F2

 

3.1 ± 6.5

7.0 ± 8.7

 

3.7 ± 5.5

4.2 ± 5.2

5.0 ± 7.4

4.9 ± 7.8

10.8 ± 16.2**

7.7 ± 13.8

Total Litter Weight (g)

F1 Day 1

F1 Day 14

F1 Day 21

F2 Day 1

F2 Day 14

F2 Day 21

 

95.1

389.5

600.0

87.3

363.2

564.2

 

89.6

362.6

567.4

101.0

384.1

599.3

 

92.4

368.8

584.8

95.6

368.9

570.4

 

88.9

343.7**

523.8**

98.0

360.0

555.2

Total number of Corpora Lutea

F0-F1

F1-F2

 

15.7 ± 1.6

15.3 ± 2.3

 

15.7 ± 2.7

17.2 ± 2.2

 

15.7 ± 1.8

16.5 ± 2.9

 

15.9 ± 2.0

16.3 ± 3.8

Total number of Implantation Sites

F0-F1

F1-F2

 

 

14.8 ± 1.7

14.4 ± 2.5

 

 

14.5 ± 2.7

16.3 ± 1.8

  

14.8 ± 2.0

15.4 ± 2.4

 

15.0 ± 1.7

16.0 ± 3.7

Total Number of Offspring Born

F0-F1

F1-F2

 

14.4 ± 2.1

13.5 ± 2.9

  

14.0 ± 2.6

15.5 ± 1.7

14.2 ± 2.4

14.6 ± 2.5

  

13.4 ± 1.7

14.8 ± 4.0

Sex ratio (% male at birth)

51.9

45.3

46.8

51.8

Table 3. Ano-Genital distance of offspring - group mean litter values.

Ano-genital distance (mm) on Day 1 post partum

Control group

Low-dose group

1500 ppm

Mid-dose group 6000 ppm

High-dose group 25000 ppm

Males

F1

F2

3.12 ± 0.34

3.01 ± 0.33

2.95* ± 0.40

3.03 ± 0.33

3.12 ± 0.46

3.01 ± 0.33

3.13 ± 0.36

3.11 ± 0.31

Females

F1

F2

1.60 ± 0.34

1.47 ± 0.19

1.65 ± 0.44

1.51± 0.24

 

1.49 ± 0.31

1.33± 0.14**

 

1.67 ± 0.34

1.49± 0.22

p** ≤ 0.01

Table 4. Nipple counts for the F1 generation at Day 11 of age.

Group

Mean Nipple count at Day 11 of age

Male

Female

Control

0.0 ± 0.0

9.4 ± 1.8

Low dose group

0.1 ± 0.5

10.4 ± 1.5

Mid-dose group

0.0 ± 0.0

9.9 ± 1.5

High-dose group

0.1 ± 0.3

10.6 ± 1.4*

p* ≤ 0.05

Conclusions:
The test substance had no effect on intrauterine development.
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Reason / purpose:
reference to same study
Reason / purpose:
read-across source
Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
- Age at study initiation: approximately 12 weeks.
- Weight at study initiation: 277-353 g (males), 180-228 g (females)
- Housing : 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). This was also applicable for Main females and Recovery animals throughout the complete treatment period. Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 (actual range: 19.7-21.9)
- Humidity (%): 40-70 (actual range: 22-71)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. The testing laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6h prior to dosing and were homogenized to a visually acceptable level.
Adjustment was made for the density of the test substance (0.944), and the specific gravity of the vehicle (1.125).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Purity: polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany)

Dose volume: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6h at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤10%) and formulations at the entire range were stable when stored at room temperature for at least 6h.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type).
Duration of treatment / exposure:
41-49 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.
Frequency of treatment:
daily, 7 days/week
Duration of test:
41-49 days
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 10-day dose range finding study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: Yes

POST-MORTEM EXAMINATIONS: Yes

For further details see Section 7.5.1
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: No

The numbers of former implantation sites and corpora lutea were recorded for all paired females.
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes, partly (see details below)
- Skeletal examinations: No
- Head examinations: No

Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made in all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Pups surviving to planned termination were killed by decapitation on lactation Day 5.
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue as well as pups from females that were killed in extremis were preserved in 10% buffered formalin for possible further examination.

Statistics:
See Section 7.5.1
Indices:
For each group the following calculations were performed:
- Mating (%): Number of females mated/Number of females paired x 100
- Fertility index (%): Number of pregnant females/Number of females paired x 100
- Conception index (%): Number of pregnant females/Number of females mated x 100
- Gestation index (%): Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100
Details on maternal toxic effects:
Maternal toxic effects:no effects
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Number of live pups: 432
Mortality: 1/119, 1/116. 1/126 and 1/71 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. This is not considered to be treatment-related, as this falls within the expected range for this strain and age.
A statistically significant lower mean number of living pups at first litter check observed at 1000 mg/kg/day was due to a low number of pups (5 in total) for one female. Since the number of pups of other females of this dose group was within the range considered normal, no toxicological relevance was ascribed to this change.
Viability index, body weights of pups, external examination for abnormalities and clinical signs of pups did not indicate any treatment-related abnormalities
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Viability index, body weights of pups, external examination for abnormalities and clinical signs of pups did not indicate any treatment-related abnormalities
Abnormalities:
not specified
Developmental effects observed:
not specified
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Reason / purpose:
read-across source
Species:
rabbit
Strain:
other: Hra:(NZW)SPF rabbits
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HRP, Inc., Denver, PA, USA
- Age at study initiation: 5.5 - 6.5 months
- Weight at study initiation: 3300 - 4446 g
- Housing: individually housed in suspended stainless steel cages
- Diet: certified rabbit diet 5322 (PMI Feeds. Inc.), ad libitum; except during dose administration
- Water: ad libitum; except during dose administration

ENVIRONMENTAL CONDITIONS
Environmental controls in the animal rooms were set to maintain temperature, relative humidity, and light/dark cycle (no further information)
Route of administration:
intravenous
Vehicle:
other: 20% lipid emulsion, no further specified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose was administered daily to rabbits via a marginal ear vein using an indwelling catheter [Insyte-W (24-gauge, 3/4-inch intravenous catheter and needle unit attached to a 30-inch extension set, Abbott Laboratories)]. Doses for rabbits were delivered using syringe pumps. The rabbits were acclimated to the dose restraint apparatus for 2.5 and 5 h on the 2 days preceding initiation of dosing.


Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Mated rabbits were received on gestation day 2 or 3 in two consecutive weekly shipments and were observed at arrival for abnormalities indicative of health problems.
Duration of treatment / exposure:
(P): 13 days during gestation
Frequency of treatment:
5 h/day, 7 days/week
Duration of test:
Day 29 of gestation / post mating
No. of animals per sex per dose:
15
Control animals:
other: 0.9% saline
Details on study design:
- Dose selection rationale: The dose rate was based on previous preclinical studies (unpublished). The 1000 mg/kg dose approximates the proposed clinical dosage. The 4280 mg/kg dose is the highest dose administered in preclinical studies that did not produce narcosis (unpublished).

- Selection of exposurre route: The intravenous route of administration was used because the lipid emulsion is intended for intravenous human administration as a component of parenteral nutrition.

- Dose volumes: 5 and 21.4 mL/kg

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily (AM and PM) for mortality and moribundity. In addition, rabbits were observed once daily for clinical observations. On dosing days, animals were observed predose, immediately (within 5 min) postdose, and approximately 1 h after completion of dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: daily and on GD 7 through 29

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Feed consumption data were collected daily beginning on GD 6.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all. Live fetuses were weighed, examined externally, then euthanatized.
- Soft tissue examinations: Yes: all. The internal organs of the rabbit fetuses were examined in the fresh state for variations and malformations, and the sex was determined. Following completion of fetal examinations, soft tissue malformations were preserved in 10% phosphate-buffered formalin.
- Skeletal examinations: Yes: all. Viscera were removed and discarded, and fetuses were processed and examined for skeletal variations and malformations; skeletal specimens were retained in glycerine.
- Head examinations: Yes: all. A mid-corona slice of the head was made to expose the internal structure of the brain for examination; the eyes were excised and examined.
Statistics:
The litter was the experimental unit for evaluation. All comparisons were made with the control group (Group 1). For rabbit data, Levene's test (Levene, 1960) was done to test for variance homogeneity. In the case of heterogeneity of variance at p « 0.05, rank transformation was used to stabilize the variance. Analysis of variance [ANOVA (Winer, 1971a)] was done on the homogeneous or transformed data. If the ANOVA was significant, Dunnett's t test (Dunnett, 1964) was used for pairwise comparisons between groups. One-way ANOVA was used to analyse body weights, body weight changes, feed consumption, and cesarcan section data. As appropriate, rabbit fetal abnormality data were analysed by the Cochran-Armitage test (Thalcur et al, 1985) for trend and departure and by the Fisher-Irwin exact test (Thalcur et al., 1985). One-way analysis of covariance [ANCOVA (Winer, 1971b)] was used to analyse fetal body weights (males, females, and combined) with the number of fetuses in the litter as the covariate. As appropriate, for values calculated to analyse litter data or mean fetal weight data, values were first derived within the litter, and the group mean values were derived as a mean of individual litter values. Group comparisons were analysed at the 5.0 and 1.0% two-tailed probability levels.
Indices:
Early and late resorptions, % fetuses dead/live, % postimplantation loss
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: at the highest dose group

Details on maternal toxic effects:
Survival was 100% for the 1000 mg lipid/kg group. One animal in the control group died on GD 11 and one animal given 4280 mg lipid/kg was sacrificed after aborting on GD 20. The animal that aborted had the lowest food consumption in the group. Decreased food consumption, resulting in a decline in the health of this animal, may have contributed to the abortion. The abortion was not considered to be test article-related as it was in the range of historical control incidence. There were no remarkable necropsy findings for either animal. Clinical observations in the test article-treated groups were limited to faecal findings (i.e., increased incidence of few or no faeces). Three animals each at 4280 mg lipid/kg had no faecal output for 1 day during the dosing period.
The data on maternal body weight changes as taken from the tabellary reporting showed a significant loss of body weights during gestational day 7 - 20 in maternal rabbits accompanied by reduced food consumption in the high dose group.
The body weight gain during GD 7-20 was found to be 95.4 g in control animals and 119.6 g in animals dosed with 1000 mg/kg bw/day, whereas at the same time animals dosed with 4280 mg/kg bw/day showed a significant loss of body weight of - 124.8 g (p < 0.01).
Food consumption in the highest dose group was only 50% of the control during GD 12 - 13 and only 10% of the control during GD19 - 20. Food consumption continued to be significantly lower for the highest dose group during the early post-treatment period (GD 20 to 24), but recovery was noted at a later interval (GD 24 to 29). The decreased food consumption observed in this study was an expected occurrence based on the high-caloric nature of the test article. There were no test article-related findings at necropsy. All pregnant animals had at least one viable foetus at scheduled caesarean section on GD 29 (i.e., no dams had total resorptions).

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Remarks:
(i.v. application)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: at the highest dose group

Details on embryotoxic / teratogenic effects:
The mean percentage of total resorptions/litter (postimplantation loss) was significantly higher and the mean percentage of live foetuses/litter was correspondingly lower in the 4280 mg lipid/kg group. Mean covariate-adjusted foetal body weights (males, females, and combined) for the 4280 mg lipid/kg group were significantly lower.
The proportion of foetuses and litters in the 4280 mg lipid/kg group with external morphological abnormalities was significantly higher than that of the control group.
The most notable findings were rachischisis and short tail seen in three and two high-dose litters, respectively. Single incidences of the following malformations were also present in the high-dose group: exencephaly, ablepharia, exophthalmus, and ectrodactyly. Single litter incidences of carpal flexure, tarsal flexure, and malpositioned shoulders were noted for the 1000 mg lipid/kg group; however, these were not considered to be test article-related. No external abnormalities were noted for the control group.
The total incidence of litters in the 1000 mg lipid/kg group with foetuses having soft tissue abnormalities was significantly higher than that of the control group; however, no significant differences were present when individual malformations and variations were analysed statistically. Although the incidence of litters in the 4280 mg lipid/kg group with viscerally abnormal foetuses was also higher than controls, the difference was not statistically significant. In general, soft tissue abnormalities were present in the test article-treated groups as single foetal or litter incidences restricted to three or four litters/group, suggesting a litter-related occurrence.
Because no soft tissue abnormalities (with the exception of absent azygous lobe of the lung) were noted for the control litters, the relationship of the abnormalities in the test article groups to the test article is inconclusive.
The proportions of foetuses with variations in the 4280 mg lipid/kg group [skull bones unossified, more than 26 presacral vertebrae, and 12 full pairs of ribs (litter incidence was also greater)] and total skeletal abnormalities were significantly higher than those in the control group. The proportions of litters in this group with foetuses having misaligned sternebrae, more than 12 full pairs of ribs, and fused ribs were also significantly higher than those in the control group.
Though not statistically significant, there was a notable increase in the number of high-dose foetuses/litter with malformations of the vertebral column. The anomalies were varied in location (cervical, thoracic, sacral, and caudal) and type (malformed, misaligned, absent, and fused). The incidence of any one of these findings was not markedly higher than that of the controls (usually only one litter was affected with a given abnormality); however, when combined, the incidence of foetuses/litters from the high-dose group with vertebral column malformations was notably higher than that of the control group.

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Remarks:
(i.v. application)
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Effect level:
4 280 mg/kg bw/day
Based on:
test mat.
Remarks:
(i.v. application)
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Cesarean section examinations of rabbits revealed higher postimplantation loss (i.e., increased incidence of resorptions) and, correspondingly, fewer live foetuses at the 4280 mg lipid/kg level. Mean foetal weights were lower for this group as well, and more litters in the group tended to have foetuses with morphological anomalies than were seen in the control group. Reduced foetal weights may have been secondary to the decreased maternal food consumption observed at this dose level.

Because treated females were consuming significantly less food than control females, the foetal effects noted for rabbits (especially the postimplantation loss and decreased foetal weights) may have been due to dietary deprivation, as opposed to a direct effect by the test article. Increased incidence of abortion and implant resorption among pregnant rabbits subjected to dietary deprivation have been reported (Matsazawa et at, 1980). There were no related patterns of major malformations for foetuses. Skeletal variations were present in all litters, including control and treated groups. Diverse abnormalities were seen that may have been associated with maternal toxicity (Khera, 1984; Manson, 1986).

Administration of the test article to rabbits at 1000 or 4280 mg lipid/kg resulted in a NOEL for developmental toxicity greater than or equal to 1000 mg lipid/kg but less than 4280 mg lipid/kg based on the adverse foetal findings (increased postimplantation loss, lower foetal body weights, and higher incidence of morphological anomalies) seen at the 4280 mg lipid/kg level. Administration of the test article at the highest dose also resulted in lower maternal food consumption and loss of body weight, therefore the observed foetal effects were probably the result of dietary deprivation, maternal toxicity or both, rather than a direct teratogenic effect of the test article.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Reason / purpose:
read-across source
Species:
rat
Strain:
other: Crl:CD BR rats
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI, USA
- Weight at study initiation: 150 - 200 g
- Housing: individually housed in suspended stainless steel cages
- Diet: Certified Rodent Chow 5002 meal (PMI Feeds, Inc.), ad libitum; except during dose administration
- Water: ad libitum; except during dose administration

ENVIRONMENTAL CONDITIONS
Environmental controls in the animal rooms were set to maintain temperature, relative humidity, and light/dark cycle
Route of administration:
intravenous
Vehicle:
other: 20% lipid emulsion was created, no further specified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose was administered daily to rats by intravenous infusion via a caudal vein using a Quik-Cath Teflon catheter connected to a syringe using an extension set. Doses for rats and rabbits were delivered using syringe pumps.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Time-mated Crl:CD BR rats were shipped by the supplier on GD 4.
Duration of treatment / exposure:
Day 6-15 of gestation
Frequency of treatment:
4 h/day, 7 days/week
Duration of test:
Day 20 of gestation / post mating
No. of animals per sex per dose:
25 or 29 females (P)
Control animals:
other: 0.9% saline
Details on study design:
- Dose selection rationale: The dose rate was based on previous preclinical studies (unpublished). The 1 g/kg dose approximates the proposed clinical dosage. The 4.28 g/kg dose is the highest dose administered in preclinical studies that did not produce narcosis (unpublished).
- Selection of exposure route: The intravenous route of administration was used because the lipid emulsion is intended for intravenous human administration as a component of parenteral nutrition. The dose was administered daily to rats by intravenous infusion for approximately 4 h/day on GD 6 through 15 via a caudal vein using a Quik-Cath Teflon catheter connected to a syringe using an extension set.
- Dose volumes: 5 and 21.4 mL/kg bw
Maternal examinations:
CLINICAL SIGNS AND CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily (AM and PM) for mortality and moribundity. On dosing days, animals were observed predose, immediately (within 5 min) postdose, and approximately 1 h after completion of dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: daily on GD 5 - 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Food consumption data were collected daily beginning on the day of receipt

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day GD 20


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: half litter; live fetuses were weighed, examined externally, sexed, then euthanatised
- Soft tissue examinations: half litter; Following completion of fetal examinations, soft tissue malformations were preserved in 10% phosphate-buffered formalin
- Skeletal examinations: half litter; Viscera were removed and discarded, and fetuses were processed and examined for skeletal variations and malformations; skeletal specimens were retained in glycerine
- Head examinations: half litter; A mid-coronal slice of the head was made to expose the internal structure of the brain for examination; the eyes were excised and examined.
Statistics:
The litter was the experimental unit for evaluation. All comparisons were made with the control group (Group 1). For rats, feed consumption, dam body weights, and fetal body weights were summarized with means and standard deviations calculated using an Excel spreadsheet program (Microsoft Corporation). For rabbit data, Levene's test (Levene, 1960) was done to test for variance homogeneity. In the case of heterogeneity of variance at p « 0.05, rank transformation was used to stabilize the variance. Analysis of variance [ANOVA (Winer, 1971a)] was done on the homogeneous or transformed data. If the ANOVA was significant, Dunnett's t test (Dunnett, 1964) was used for pairwise comparisons between groups. One-way ANOVA was used to analyze body weights, body weight changes, feed consumption, and cesarcan section data. As appropriate, rat and rabbit fetal abnormality data were analyzed by the Cochran-Armitage test (Thalcur et al, 1985) for trend and departure and by the Fisher-Irwin exact test (Thalcur et al., 1985). One-way analysis of covariance [ANCOVA (Winer, 1971b)] was used to analyze fetal body weights (males, females, and combined) with the number of fetuses in the litter as the covariate. As appropriate, for values calculated to analyze litter data or mean fetal weight data, values were first derived within the litter, and the group mean values were derived as a mean of individual litter values. Group comparisons were analyzed at the 5.0 and 1.0% two-tailed probability levels.
Indices:
early and late resorptions, % fetuses dead/live, % postimplantation loss
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: effects in the highest dose group

Details on maternal toxic effects:
The only test article-related findings observed were associated with tail lesions in both test article groups and the occasional occurrence of red tinged urine (8 of 29) and vaginal bleeding (1 of 29) in the high-dose group. The tail findings were predominantly those of discoloration and ulceration. Incidences were 1/25, 14/25, and 23/29 for these tail lesions in the control, low-, and high-dose groups, respectively, and ranged from mild to severe with some necrosis and partial loss of the tail.
These findings were generally considered to be related to occurrences of observed extravasation of the MCT:LCT lipid test article into perivascular areas. Evaluation of urine collected from one high-dose animal suggested a bacterial infection of the urogenital tract. Clinical observations for this animal included occasional red-tinged urine and vaginal bleeding. This rat was noted as having a large urinary bladder stone and kidney hydronephrosis at necropsy.
There were no marked differences in mean body weights or feed consumption for the low-dose group compared with those of the control group. However, the high-dose group consistently exhibited lower body weights beginning 1 day after dose administration throughout the remainder of the study. Feed consumption was also notably lower for nine of the ten days during dosing, with an increase in feed consumption after completion of dose administration (GD 15). The decrease in feed consumption at the high-dose level was expected based on the high-caloric nature of the test article.
Necropsy findings were primarily related to tail effects and were observed for most rats in the high-dose group and some rats in the low-dose group. In addition to tail effects, there was a trend toward an increasing incidence of necropsy findings in the high-dose group, including enlarged lymph nodes, enlarged spleen, hydronephrosis/enlarged renal pelvis, small thymus, and small red lung foci. These changes indicated that the high-dose group was likely exhibiting test article effects.
There was a slight trend toward decreasing mean gravid uterine weights in proportion to increasing test article dose; however, due to the large variability between groups, group mean uterine weights appeared to be similar. With the exception of one control dam, all females were pregnant and had at least one viable fetus/litter (i.e., no dams had total resorptions).
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Remarks:
(i.v. application)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no significant group differences in preimplantation or postimplantation loss or in the mean percentage of live or resorbed fetuses; no dead fetuses were present. Mean fetal sex ratios of the test article-treated groups were comparable with those of controls. There were no apparent effects on mean fetal body weight (combined, males, or females). There were no test article-related fetal external, soft tissue, or skeletal observations.
A high incidence of folded retina in control and test article-treated groups was attributed to shrinkage of the retina during storage in alcohol prior to being transferred to Bouin's fixative.
Omphalocele and cleft palate were observed in one fetus each in the control and high-dose groups, respectively. The only fetal skeletal malformation observed (malformed/misshapen skull bones) was present in one fetus each from two control litters. Fetal skeletal variations were present in control and test article-treated groups in a nondose-related pattern.

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
4 280 mg/kg bw/day
Based on:
test mat.
Remarks:
(i.v. application)
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Upon intravenous application of 4280 mg/kg bw/day during gestational Days 6 - 15, medium chain triglycerides exerted no teratogenic effects in rats.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to the endpoint discussion for further details). Taken together, the information from these independent sources is consistent and provides sufficient weight of evidence for hazard assessment leading to an endpoint conclusion in accordance with Annex XI, 1.2, of Regulation (EC) No 1907/2006. Therefore, the available information as a whole is sufficient to fulfil the standard information requirements set out in Annex VIII-X, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for read-across

There are no data on the developmental toxicity of glycerides, C16-18 and C18-unsaturated mono-and di-citrates (CAS 91052-16-3). The assessment was therefore based on studies conducted with analogue substances as part of a read-across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13) and within Chapter 5.1 of the CSR.

CAS No. 736150-63-3

A Two-generation reproduction toxicity and developmental neurotoxicity study with glycerides, castor-oil, mono, hydrogenated, acetates was performed in Sprague-Dawley Crl:CD®(SD) IGS BR rats according to OECD guidelines 416 and 426 and in compliance with GLP (Fulcher, 2011). Groups of 28 parental animals per sex were exposed daily to the test substance at dietary concentrations of 1500, 6000 and 25000 ppm, corresponding to mean achieved dose levels of 82, 324 and 1159 mg/kg bw/day in males and mean achieved dose levels of 146, 587 and 2200 mg/kg bw/day in females, respectively. Parental males were treated with the test substance 10 weeks during maturation and throughout mating, gestation and until completion of the parental female lactation phase. Parental females received the test substance 10 weeks during maturation and throughout mating, gestation and until Day 21 of the lactation phases. After weaning, groups of 24 animals per sex of the F1 generation were exposed daily to the same dietary concentrations as their parental animals. The corresponding mean achieved dose levels in these animals were 109, 435 and 1342 mg/kg bw/day for F1 males and 160, 630 and 2262 mg/kg bw/day for F1 females, respectively. Males of the F1 generation received the diets a minimum of 10 weeks during maturation and subsequently throughout mating, gestation and until completion of the F1 female lactation phases. Females of the F1 generation were treated a minimum of 10 weeks during maturation and subsequently throughout mating, gestation and lactation phases. Between weaning and the formal start of the F1 generation, the animals continued to receive their appropriate treated diet. Animals of the F2 generation were exposed to the diets from weaning (Day 21 of age) to termination of the study on Day 70 of age. In each generation, animals administered with the high dose initially received the test material at a concentration of 15000 ppm and this was increased to 20000 ppm and finally 25000 ppm as the study progressed. Similar constituted control groups for each generation received laboratory diet enriched with Arachis oil to ensure comparable caloric intakes.

In animals of the F1 and F2 generation, no effects on viability and no clinical signs were observed either as consequence of maternal treatment after birth or due to of direct treatment after weaning.

Body weights and food consumption in both generations of offspring were not adversely affected by treatment with the test substance. There were no obvious effects of treatment on the sexual maturation of either sex for the F1 animals receiving the test substance. Sex ratio at birth and subsequently at Day 21 of age was similar to concurrent control in all treatment groups over both generations. The slight variations in the mean ano-genital distance which were observed on Day 1 in F1 males at 1500 ppm and F2 females at 6000 ppm as well as the marginally higher visible nipple counts in F1 females at 25000 ppm were considered to be incidental and not treatment-related.

In both generations, organ weights were not adversely altered by exposure to the test substance. Non-dose-related variations in absolute and relative organ weights were observed in spleen and uterus of females of both generations.

No effects were observed on gross pathology in offspring and there were no histopathological findings observed for male and female behavioural offspring.

The neurotoxicological examination of the F0-F1 offspring of treated animals did not reveal any treatment-related effects when compared to the current F0-F1 offspring of the untreated control animals.

Based on the results of the study, the NOAEL for developmental toxicity in male and female rats of the F1 and F2 generation was 1342 and 2262 mg/kg bw/day, respectively. These doses corresponded to a concentration of 25000 ppm of the test substance in the diet.

Short-, medium- and long-chain triglycerides (SCT, MCT, LCT)

In a prenatal developmental toxicity study performed similar to OECD guideline 414, the effects of medium Chain Triglycerides (MCT) on female Crl:CD BR rats were investigated during Days 6 to 15 of gestation (Henwood, 1997). The animals (25 or 29 for the low and high dose, respectively) received the test substance in 20% emulsion containing a 3:1 ratio of MCT:LCT (Long Chain Triglycerides) at doses of 1000 and 4280 mg/kg bw/d by intravenous infusion via the caudal vein for a 4-h period per day. A control group of 25 animals received 0.9% saline. On Day 20 of gestation, dams were sacrificed and maternal as well as foetal examinations were performed. At 4280 mg/kg bw/day, maternal toxicity occurred and involved an increased incidence of necropsy findings on the tail, which was considered to be due to extravasation of the MCT:LCT lipid test article into perivascular areas. In addition to tail effects, there was a trend toward an increasing incidence of necropsy findings in the high-dose group, including enlarged lymph nodes, enlarged spleen, hydronephrosis/enlarged renal pelvis, small thymus, and small red lung foci. There were no significant group differences in pre-implantation or post-implantation loss or in the mean percentage of live or resorbed foetuses in treated animals. No dead foetuses were found and the mean foetal sex ratios and the mean foetal body weight of the treated animals were comparable to those of controls. No test substance-related external, soft tissue, or skeletal malformations were noted in the foetuses of exposed mothers. Based on the results of the study, the NOAEL for developmental toxicity in male and female Crl:CD BR rats was established at ≥ 4280 mg/kg bw/day.

In the same study, 15 female Hra:(NZW)SPF rabbits were treated daily with MCT (Medium Chain Triglycerides) by a 5-h intravenous infusion via a marginal ear vein at doses of 1000 and 4280 mg/kg bw/d from gestation day 6 through 19 (Henwood, 1997). A similar constituted control group was injected with 0.9% saline. Administration of the test substance resulted in lower maternal food consumption and significant body weight loss during treatment at 4280 mg/kg bw/day. All pregnant animals had at least one viable foetus at scheduled caesarean section on GD 29. The observed foetal effects (i.e. increased resorptions, decreased foetal body weights, and increased incidence of morphological anomalies) were assumed to be the result of dietary deprivation, maternal toxicity, or both, rather than a direct teratogenic effect of the test article. Based on these results, the NOAEL for developmental toxicity was set at 1000 mg/kg bw/day in rabbits.

Conclusion for developmental toxicity

A read-across approach was applied to assess the developmental toxicity potential of the target substance glycerides, C16-18 and C18-unsaturated mono-and di-citrates (CAS 91052-16-3). The studies on the structural analogous substances, glycerides, castor-oil, mono, hydrogenated, acetates (CAS 736150-63-3), glycerides, C8-18 and C18-unsatd, mono- and di-, acetates (CAS 91052-13-0)  and Short-, medium- and long-chain triglycerides (SCT, MCT, LCT) did not show any treatment-related effects up to the highest tested dose level. Therefore, as the available data did not identify any hazard for developmental toxicity, glycerides, C16-18 and C18-unsaturated mono-and di-citrates (CAS 91052-16-3) is considered to have no toxic effects to fetal development. 


Justification for selection of Effect on developmental toxicity: via oral route:
Hazard assessment is conducted by means of read-across from structural analogues. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between the source and target substances and overall quality assessment (refer to the endpoint discussion for further details).

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to glycerides, C16-18 and C18-unsaturated mono-and di-citrates (CAS 91052-16-3) data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Based on the analogue read-across approach, the available data on toxicity to reproduction and developmental toxicity does not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and is therefore conclusive but not sufficient for classification.