Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
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. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance. Justification for grouping of substances and read-across: The study is selected as a suitable study for the assessment of potential reproduction toxicity effects within the Glycerides category. Read-across from Glycerides, castor-oil-mono, hydrogenated, acetates (main component: 12-acetoxy-octadecanoic acid (2,3-diacetoxy)propyl ester [CAS 330198-91-9]) (CAS No. 736150-63-3) 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).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

open allclose all
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)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:
The test substance had no effect on reproductive performance.