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Key value for chemical safety assessment

Effects on fertility

Description of key information

The assessment is based on the data currently available. New studies, based on the category review and the final decisions issued for some of the category substances, which are also relevant for this assessment, are currently being conducted. The hazard assessment with respect to toxicity to reproduction will be updated once all ongoing studies have been finalised.

OECD 416, rat, 2 gen, oral: not toxic to reproduction
NOAELrep = 300 mg/kg bw/day; LOAELrep > 300 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Jun 1996 - 20 Mar 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)BR (VAF plus)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd., England
- Age at study initiation: 4-5 weeks
- Weight at order: 120 - 150 g
- Age after acclimatisation: males 6-7 weeks, females 14-15 weeks: Mating of F1 animals started at an age of 16 weeks
- Fasting period before study: No
- Housing: Group housed, except for mating period. During pregnancy and lactation period females were housed individually.
- Diet: Pelleted SQC rat and mouse No. 3 Breeder; ad libitum
- Water: Purified water (UHP, equivalent to single distilled water) produced by reverse osmosis of mains water in an 'Elgastat Prima' water purification unit was provided in high-density polycarbonate bottles fitted with stainless steel tops and sipper tubes; ad libitum
- Acclimation period: males 12 days, females 6 - 7 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 27
- Humidity (%): 36 - 62
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
The test article was formulated for dosing as solutions in the vehicle, purified drinking water. For formulation, the weighed quantity of test article was mixed with the appropriate volume of vehicle. Separate formulations were prepared for each dose level. Formulations were prepared once weekly and stored in polycarbonate aspirators at ambient temperature in the animal room during the week of use.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Maximum 14 days
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged: Individually
- Any other deviations from standard protocol: Due to reduced numbers of litters in each group of the F1a generation the F0 generation was re-paired following weaning of the F1a generation to produce a F1b generation. Females were allowed to rear their offspring to weaning on day 21 post partum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Two 10 mL samples from the middle of the aspirator of each dose group prepared for weeks 1, 5, 9, 13, 17, 21, 25, 29 and 33 have been quantified via HPLC analysis using an internal standard for quantification. The actual concentrations proved the nominal concentrations.
Duration of treatment / exposure:
P males: 11 weeks before mating, during both mating periods (for F1a and F1b), until necropsy (total 198 days).
P females: 2 weeks before mating, during both mating periods (for F1a and F1b), pregnancy and lactation, until necropsy (Day 21 post partum) (total 71 days for F0 to F1a and 114 days for F0 to F1b).
F1 males were dosed from birth until approximately 16 weeks of age, during mating, until necropsy (total 110 days).
F1 females were dosed from birth until approximately 16 weeks of age, during mating, pregnancy and lactation period (total 119 days).
Frequency of treatment:
Daily
Details on study schedule:
F1 parental animals were mated 16 weeks after selected from the F1 litters.
Dose / conc.:
0.03 other: % in water
Remarks:
concentration proven by analytical examination; corresponding to ~ 30 mg/kg bw/day (rough estimate, actual values are rather higher). For details on the exact calculated doses, please refer to the result section for water consumption.
Dose / conc.:
0.1 other: % in water
Remarks:
concentration proven by analytical examination; corresponding to 100 mg/kg bw/day (rough estimate, actual values are rather higher). For details on the exact calculated doses, please refer to the result section for water consumption.
Dose / conc.:
0.3 other: % in water
Remarks:
concentration proven by analytical examination; corresponding to 300 mg/kg bw/day (rough estimate, actual values are rather higher). For details on the exact calculated doses, please refer to the result section for water consumption.
Dose / conc.:
0.015 other: % in water
Remarks:
Females of group 2 during lactation
Dose / conc.:
0.05 other: % in water
Remarks:
Females of group 3 during lactation
Dose / conc.:
0.15 other: % in water
Remarks:
Females of group 4 during lactation
No. of animals per sex per dose:
30 (F0 gen), 25 (F1 gen)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Due to the increase in water consumption normally observed in lactating rats, females received drinking water at concentrations of 0.015, 0.05 or 0.15% during the period of lactation (groups 2, 3 or 4, respectively) to maintain a more constant intake relative to body weight.
- Fasting period before blood sampling for clinical biochemistry: no
Positive control:
Not required.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Twice daily for mortalities and once daily for clinical signs of toxicity

BODY WEIGHT: Yes
Males: at weekly intervals. Females weekly during pre-mating, on days 0, 7, 14 and 20 of pregnancy and on days 0, 4, 7, 14, 21 post partum.

FOOD CONSUMPTION: Yes
At weekly intervals. Additionally for females from days 1-4, 4-7, 7-11 and 11-14 post partum

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
Daily for males and females during pre-mating, for males at the end of the mating period and for females during pregnancy and lactation. Water consumption was not measured for mating pairs.

OTHER:
For haematology and clinical chemistry evaluations, blood samples were taken from 10 males and 10 females of each dose group one week prior the F0-F1a and the F1-F2 mating period. Following parameter were assessed: leucocyte differential count, total leucocyte count, A/G ratio, alanine aminotransferase, albumin, alkaline phosphatase, aspartate aminotransferase, blood urea nitrogen, creatinine, globulin, total bilirubin, total protein, and triglycerides.
Oestrous cyclicity (parental animals):
Beginning two weeks before the start of the first F0 and the F1 mating period and during all the mating periods until confirmation of maing or end of the relevant periods vaginal smears were taken daily and examined for estrous cycle stage.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, sperm count in epididymides, percent motile sperms (incl. calculation of straight line velocity and average pathe velocity), sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, body weight and body weight gain, physical or behavioural abnormalities, time until developmental milestones are reached (e.g. ear opening, balanopreputial separation of males, vaginal perforation of femals, etc.)

FUNCTIONAL OBSERVATIONS
On day 22 post partum the auditory startle habituation response was evaluated using the SR-Screening System on pups selected for special necropsy. At 28 days of age the selected pups were examined for learning potential using a water filled e-maze in two session on consecutive days. A session consisted of six runs. At approximately 28-35 days post partum the selected F1-generation pups were tested for motor activity.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead. Necropsy was performed on all pups killed prematurely or found dead and for surplus pups after selection for special necropsy and rearing
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after the mating period
- Maternal animals: All surviving animals after the last litter of each generation was weaned.

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

ORGAN WEIGHTS
Following organs were weighed: brain, pituitary gland, liver, adrenals, spleen, thymus, kidneys, testes, epididymides (toatl and cauda), seminal vesicles, prostate gland, coagulating gland, ovaries, oviducts, uterus, cervix, vagina

HISTOPATHOLOGY
The following tissues were prepared for microscopic examination: adrenals, aorta, brain, caecum, colon, duodenum, epididymides, femur, ileum, jejunum, kidneys, liver, mesenteric lymph node, oesophagus, ovaries, pituitary, prostate, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, stomach, submandibular lymph node, testes, thymus, thyroids, uterus, vagina, gross lessions.
Detailed evaluation were further performed on opened stomach to further examine the glandular and non-glandular regions of the stomach for possible signs of irritation.
Postmortem examinations (offspring):
SACRIFICE
Necropsy was conducted on all pups killed prematurely or found dead and for surplus pups after selection for special necropsy and rearing.

GROSS NECROPSY
- One male and one female pup from each of the first twenty litters comprising sufficient pups were selected from F1a and F2 litters for special necropsy. Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Special necropsy comprised assessment of organ weights and fixation in neutral buffered formaldehyde.

ORGAN WEIGTHS
The following tissues were weighed during special necropsy: brain, liver, adrenals, spleen, thymus, kidneys, testes, ovaries.

HISTOPATHOLOGY
The following tissues were prepared for microscopic examination of the F1 generation: adrenals, aorta, brain, caecum, colon, duodenum, epididymides, femur, ileum, jejunum, kidneys, liver, mesenteric lymph node, oesophagus, ovaries, pituitary, prostate, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, stomach, submandibular lymph node, testes, thymus, thyroids, uterus, vagina, gross lessions.
Statistics:
Group means and standard deviations were calculated for each observation time. Analysis of variance (ANOVA) was performed on all parameters. Heterogeneity of variance was analysed using Levenes test. Williams test was performed to compare high dose with control at the two-sided 5% level. Kruskal-Wallis ANOVA was performed to assess overall differences between treatment groups followed by Shirley´s test.
Reproductive indices:
Copulation index = (number of animals mated) / (number of animals paired) x 100
Fertility index = (number of animals fertile) / (number of animals mated) x 100
Gestation index = (number of pregnant females with live born pups) / (number of pregnant females) x 100
Offspring viability indices:
Live birth index = (number of pups born alive) / (total number of pups born) x 100
Viability index 1 = (number of pups alive on day 4 post-partum before culling) / (number of pups born alive) x 100
Viability index 2 = (number of pups alive on day 7 post-partum) / (number of pups alive on day 4 post-partum after culling) x 100
Viability index 3 = (number of pups alive on day 14 post-partum) / (number of pups alive on day 7 post-partum) x 100
Viability index 4 = (number of pups alive on day 21 post-partum) / (number of pups alive on day 14 post-partum) x 100
Cumulative survival index = (number of pups alive on day 21 post-partum) / (number of pups alive on day 4 post-partum) x (number of pups alive on day 4 post-partum before culling) / (total number of pups born) x 100
Sex ratio = ((number of male pups born) / (total number of pups born) x 100) / ((number of female pups born) / (total number of pups born) x 100)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Males:
No test item related clinical signs of toxicity were observed. Observations that were noted were minor in nature and were of a type and frequency that is considered to be normal for animals of this age and strain. One male at 0.3% was noted to have a sunken left eye on Day 84 on. At necropsy, this eye was noted to be small. Histologic examination revealed traumatic disruption of the eye. It was considered not to be an effect of treatment.
A small number of animals in each group were noted to be aggressive. This finding did not show a dose-related distribution and was apparent in the control group, thereby precluding an effect of treatment.
Females:
There were no clinical observations noted that were considered to be related to treatment. Observations that were noted were minor in nature and were of a type and frequency that is considered to be normal for animals of this age and strain.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Males:
One male at 0.03% was killed in extremis on Day 21 due to weight loss, teeth abnormalities, mouth lesion, swollen nose and noisy breathing (caused by accidental trauma). There were no factors contributory to death found at necropsy. The observed effects were considered not treatment-related.
One male at 0.1% was found dead on Day 37. Prior to death, this animal had been noted with teeth abnormalities. There were no factors contributory to death found at necropsy and therefore, this death was considered incidental and unrelated to treatment.
Females:
2, 2, 3 and 5 females were found dead or were killed prematurely at control, 0.03, 0.1 and 0.3%, respectively. Although the incidences in the groups treated at 0.1 and 0.3% were higher than in the other groups the observations and timings of these losses do not support any relationship to dosage and mortalities are considered to be not treatment related. For details on mortalities, please refer to Attachment 1.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 0.3% treatment groups, there was a statistically significant and treatment-related reduction in body weights of males and females during the first two weeks of the study (p<0.01 in the first week). Thereafter, mean body weight gain in this group was generally similar to that of the control group with the exception of Week 18 and 19 where a statistically significant (p<0.05) reduction in body weight gain was observed. This group showed a reduction in water consumption but not in food consumption. Therefore, the reduction in body weight gain may be related to palatability of the drinking water and may not be a direct effect of treatment on body weight.
During pre-mating, as a response to the initial reduction in body weight gain mentioned before, there was a compensatory statistically significant (p<0.05 - 0.01) increase in body weight gain in females at all dose levels during the second week of treatment.
During the second pre-mating dosing period (Days 64 - 71), mean body weight gain in females of the treated groups was generally higher than that of the control group. The difference achieved statistical significance in the 0.3% group. This was considered to be due to unusually high values for three individuals.
During pregnancy (P0 - F1a generation) there was no effect of treatment on female body weights. During lactation, body weight gain in the latter half of lactation of the first litter (Days 14 - 21) were slightly lower at 0.3% than those of the control group, which was due to three females with unusually low values in this period. Therefore, it is concluded that generally, there was no effect of treatment on maternal body wight and body weight gain in this period.
For the pregnancy period of P0 - F1b, in groups treated at 0.1 and 0.3% there was a slight reduction in body weight gain over Days 7 - 20 of pregnancy, but it was not statistically significant. During lactation, there was generally no effect on maternal body weight gain. In the 0.1% group, mean values over Days 0 - 4 of lactation were lower than those of the control group, but it was due to the low value for one female and therefore considered coincidental and unrelated to treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no effect of treatment at any dose level on male food consumption.
In contrast, in females during pre-mating, there was a treatment-related reduction in mean food consumption during Day 1 - 15. The difference was most marked and statistically significant (p<0.05) in the 0.3% group in the first week (Days 1 - 8). This reduction was considered to be attributable to the reduced water intake (due to palatability of the formulated drinking water) due to the dry nature of the diet and hence was not an adverse effect of the treatment. During the second pre-mating period (Days 64 - 71), mean food consumption in the treated groups was higher than that of the control group (not statistically significant. During pregnancies (both for F1a and F1b) and lactation, there was no effect on food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
In males and females during premating there was a treatment-related decrease in water consumption in the group treated at 0.3%, with mean values being statistically significantly reduced (p<0.01 - p<0.05) throughout the P0 generation phase (males) or during Weeks 1 - 3 (females).The reduction was possibly due to the palatability of the formulated drinking water. There was no effect of treatment on mean water consumption in the 0.03 and 0.1% group in males and females, and values were generally higher than those of the control.
In females during the second pre-mating phase, water consumption was, however, comparable to controls. During pregnancy (P0 - F1a generation), there was a treatment-related reduction in mean maternal water consumption at 0.3% (p<0.05 over Days 7 - 14 of pregnancy). Again, it was considered related to palatability. Similar effects occurred during lactation of that generation (Days 0-7 being statistically significant). It should also be noted that during this period, females treated at 0.03 and 0.1% had higher water consumption than controls.
In females during pregnancy of the P0 - F1b generation, there was a treatment-related reduction in mean maternal water consumption for the dosing groups 0.1 and 0.3%, with the difference being most marked at 0.3% during Days 7 - 14 (p<0.05) and 14 - 20 (p<0.001). At 0.1%, difference were slight up to Day 14 of pregnancy but achieved statistical significance over Days 14 - 20 (p<0.05). The effect was considered unrelated to treatment at this dose level, as it was not observed in the P0-F1a and P1-F2 generations. It should be noted that for females at 0.03% during pregnancy, mean maternal water consumption was not affected by treatment, but higher than those of the control. Similar effects were noted during lactation, but differences were slight and not dose-related or statistically significant. The effects were considered treatment-related for females at 0.3% only, but values were also different at 0.1 (reduced) and 0.03% (increased).

Achieved doses (for the 0.03, 0.1 and 0.3% dosing groups, respectively):
P0 males: 27.4, 86.6 and 222.9 mg/kg bw/day
P0 - F1a females: 39.0, 120.9 and 316.2 mg/kg bw/day
P0 - F1b females: 38.8, 117.2 and 337.9 mg/kg bw/day
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 0.1 and 0.3%, reduced triglyceride levels in females was observed. The values were however within the range of the historical control data (see Attachment 2), and therefore, the effect was considered unlikely to be the result of treatment with the test substance.
Endocrine findings:
not specified
Description (incidence and severity):
The following parameters related to endocrine disruption were evaluated:
- Organ weight of: brain, pituitary gland, liver, adrenals, testes, epididymides (total and cauda), seminal vesicles, prostate gland, coagulating gland, ovaries, and uterus.
- Histopathology of: adrenals, brain, epididymides,ovaries, pituitary, prostate, seminal vesicles, testes, thyroids (incl. parathyroids), uterus, and vagina
- Sperm and estrous cyclicity examinations
- Learning and development
- Copulation index, fertility index, gestation index
- Viability index, live birth index, survival index and sex ratio
- Sexual development in offspring (age at balano-preputial separation; age of vaginal perforation)
For details, please refer to the respective result section.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A slightly decreased incidence and degree of centrilobular hepatocyte hypertrophy was observed in males..... (tbc)
Two animals had morphological alterations in the stomach which were further investigated (tbc).
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Semen analysis revealed slight but significantly reduced straight line velocity (VSL) of the sperm at 0.3% (19.5 ± 8.8 compared to 25.1 ± 10.9 µm/s; p<0.05) and 0.1% (p<0.05). However, all values remained within background range (see Attachment 4).
In contrast, no treatment-related effect on sperm morphology and motility were observed. There was also no effect on sperm concentration or average path velocity (VAP), and generally, all values were within background range.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect on time to mating, number of animals mated, or copulation indices. The fertility index was low in all groups (incl. controls) both at the P0-F1a and P0-F1b generations. There was no clear effect of treatment, but differences were most marked at 0.3%. However, as there was no effect on fertility in the P1-F2 generation, the observed effect was considered likely to be fortuitous.

In the P0-F1a generation, pregnancy parameters, i.e. duration of gestation, gestation index, pup survival from Day 4 - 21 post-partum (survival indices 2, 3 and 4), live birth index and mean number of pups born, were unaffected by treatment. Pup survival from Day 0 - 4 post-partum (viability index 1) and cumulative survival index were slightly lower in treated groups compared to controls. However, the differences were slight, not statistically significant and did not follow a dose-related pattern, and therefore, these effects were considered unrelated to treatment.
In the P0-F1b generation, duration of gestation, gestation index, viability indices 1 - 4, cumulative survival index and mean number of pups born were unaffected by treatment. At 0.03%, mean live birth index was slightly lower (not statistically significant), and was considered to be due to a low value for one female in this group, and at the other dose groups, mean live birth index was similar to controls.
BODY WEIGHTS, FOOD CONSUMPTION AND WATER CONSUMPTION
The effects observed were considered related to treatment, but not an adverse effect of the test substance itself. The effects were considered to be attributable to the palatability of drinking water formulations with the test substance. It is likely that palatability issues impacted water consumption and subsequently food consumption as well due to the dry nature of the diet provided. Consequently body weights were also affected.

IMMUNOTOXICITY
Possible effects on haematological and clinical biochemistry parameters were within the range of HCD and therefore, it was concluded that these effects were unlikely to be attributable to treatment with the test substance.

ORGAN WEIGHTS:
In F1 animals, there was no clear dose-response relationship detected in the liver weight changes of the parental generations. No influence on liver weight development was seen in the F2 generation. None of the groups revealed any histopathological or clinical-chemical findings, which could be attributed to hepatotoxicity. This led to the conclusion that this untypical liver weight reduction was of no toxicological relevance (non-adverse).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test item related effects on the copulation index was observed. The fertility index was low in each group of the P0 generation. Consequently F0 females were paired after weaning of F1a generation with a different male from the original pairing. The fertility index at 0.3% was slightly decreased compare to the other groups for P0-F1b females. No effect on fertility was observed in the P1-F2 generation. Thus this finding was considered to be not treatment related. No treatment related effect on duration of gestation and gestation index were observed.

SPERM PARAMETERS
It was concluded that the effects seen in VSL were considered unrelated to treatment as values were within the HCD range and no other sperm parameters (morphology, concentration, motility, VAP) were adversely affected.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No test item related effects were observed, except for slight signs of local irritation in the stomach at the top dose.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
0.3 other: %
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related adverse effects observed up to and including the highest dose tested.
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Dose corresponding to 222.9 mg/kg bw/day in males and 316.2 -337.9 mg/kg bw/day in females.
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
0.3 other: %
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related adverse effects observed up to and including the highest dose tested.
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Dose corresponding to 222.9 mg/kg bw/day in males and 316.2 -337.9 mg/kg bw/day in females.
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no observations that were considered to be related to treatment. Observations that were noted were minor in nature and were of a type and frequency that can occur spontaneously in this strain of animals. There was one isolated incident of convulsions in a male at 0.03%. It was considered a chance occurrence unrelated to treatment and the animal recovered.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was only one case of mortality in a control male, which was euthanized due to health problems caused by teeth abnormalities. One control female was killed in extremis due to symptoms of hypoactivity with slow breathing, pallor, piloerection, vaginal discharge and reduced faecal output. One further female at 0.03 was killed in extremis on Day 111 after being observed emaciated and hypoactive, with hunched posture, partially closed eyes and reduced faecal output. This animal was pregnant and did litter but the entire litter was dead or killed in extremis by Day 2 of lactation. The litter observations show evidence of a lack of maternal care. In view of this distribution of these findings, the deterioration in clinical condition of these females and letter was considered to be coincidental and unrelated to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During premating there was no effect on body weight and body weight gain in males and females.
There was a slight reduction in body weight gain during the P1 - F2 generation pregnancy at 0.1% and 0.3% over Days 14 - 20 of pregnancy. During lactation, there was a slight increase in maternal body weight loss over Days 14 - 21 of lactation (body weight loss in this period is a normal response). The difference was most marked in the 0.3% treatment group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no effect of treatment at any dose level on male food consumption.
In females during pre-mating, there was no adverse effect observed. The mean values were generally higher than those of the control, and statistically significant for the 0.3% group (p<0.05) over days 50 - 57.
During pregnancy, there was no effect on maternal food consumption. During lactation, values in the treated groups were higher than those of the control group throughout but did not follow a dose-related pattern and were therefore not considered a treatment-related effect.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
In males, there was a treatment-related decrease in water consumption at 0.3% possibly due to the palatability of the formulated drinking water from Week 4 on. The difference was statistically significant (p<0.01 - p<0.05). Between Week 1 - 4, mean water consumption was comparable to controls.
In females during premating, there was also a treatment-related reduction in mean water consumption at 0.3% (p<0.01 - p<0.05). Values at 0.1 and 0.03% were higher than those of the control, but it was not considered an effect of the treatment. During pregnancy and lactation, similar effects were observed, but during lactation, these effects were not statistically significant.

Achieved doses (for the 0.03, 0.1 and 0.3% dosing groups, respectively):
P1 males: 38.6, 126.0 and 333.5 mg/kg bw/day
P1 - F2 females: 45.0, 149.5 and 369.4 mg/kg bw/day.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
For males at 0.1 and 0.3%, there was a small increase in neutrophil levels with a corresponding reduction in lymphocyte levels. As the values were within the historical control data range (see Attachment 3), they were considered unlikely to be the result of treatment with the test substance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes in biochemistry parameters were observed. There was a slight increase in total protein levels for all treated female groups. Although the differences were statistically significant, they did not follow a dose-related pattern and so this was considered to be coincidental and unrelated to treatment. There was also a slight increase in albumin levels for females at 0.3% which was statistically significant but the change was considered too small to be of toxicological significance (see Attachment 3).
Endocrine findings:
not specified
Description (incidence and severity):
The following parameters related to endocrine disruption were evaluated:
- Organ weight of: brain, pituitary gland, liver, adrenals, testes, epididymides (total and cauda), seminal vesicles, prostate gland, coagulating gland, ovaries, and uterus.
- Histopathology of: adrenals, brain, epididymides,ovaries, pituitary, prostate, seminal vesicles, testes, thyroids (incl. parathyroids), uterus, and vagina
- Sperm and estrous cyclicity examinations
- Learning and development
- Copulation index, fertility index, gestation index
- Viability index, live birth index, survival index and sex ratio
- Sexual development in offspring (age at balano-preputial separation; age of vaginal perforation)
For details, please refer to the respective result section.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "haematology" and "clinical biochemistry findings" sections for details.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In males, at 0.3% and 0.1%, a treatment-related and statistically significant reduction in absolute (p<0.05), body weight-related (p<0.01 and p<0.05, respectively), and brain-related (p<0.05) liver weights, were observed for males. The dose-response was not very clear.
In females, decreases were seen for absolute and relative brain and adrenal weights (p<0.05), but it was considered to be a result of body weight increases and not a direct treatment-related effect. Other changes were considered incidental.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A slightly decreased incidence and degree of centrilobular hepatocyte hypertrophy was observed.,.... (tbc)
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no effect on time to mating. Fertility indices and copulation indices were similar to those of the control group. Mean values for duration of gestation, gestation index, viability indices 1-4, cumulative survival index and mean number of pups born was similar between treated and control groups.
BODY WEIGHTS, FOOD CONSUMPTION AND WATER CONSUMPTION
The effects observed were considered related to treatment, but not an adverse effect of the test substance itself. The effects were considered to be attributable to the palatability of drinking water formulations with the test substance. It is likely that palatability issues impacted water consumption and subsequently food consumption as well due to the dry nature of the diet provided. Consequently body weights were also affected.

IMMUNOTOXICITY
Possible effects on haematological and clinical biochemistry parameters were within the range of HCD and therefore, it was concluded that these effects were unlikely to be attributable to treatment with the test substance.

ORGAN WEIGHTS:
Generally, there was no clear dose-response relationship detected in the liver weight changes of the parental generations. No influence on liver weight development was seen in the F2 generation. None of the groups revealed any histopathological or clinical-chemical findings, which could be attributed to hepatotoxicity. This led to the conclusion that this untypical liver weight reduction was of no toxicological relevance (non-adverse).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test item related effects on the copulation index was observed. The fertility index was low in each group of the P0 generation. Consequently F0 females were paired after weaning of F1a generation with a different male from the original pairing. The fertility index at 0.3% was slightly decreased compare to the other groups for P0-F1b females. No effect on fertility was observed in the P1-F2 generation. Thus this finding was considered to be not treatment related. No treatment related effect on duration of gestation and gestation index were observed.


HISTOPATHOLOGY (PARENTAL ANIMALS)
No test item related effects were observed, except for slight signs of local irritation in the stomach at the top dose.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
0.3 other: %
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to and including the highest dose tested.
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Dose corresponding to 333.5 mg/kg bw/day in males and 369.4 mg/kg bw/day in females.
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
0.3 other: %
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to and including the highest dose tested.
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Dose corresponding to 333.5 mg/kg bw/day in males and 369.4 mg/kg bw/day in females.
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F1a:
No effects on pup survival (day 4 - 21 post partum), live birth index and mean litter size were observed. Pup survival of the F1a generation (day 0 - 4 post partum) was slightly lower in the treated groups when compared to control. The differences were not significant, showed no does response relationship and were not observed for F1b and F2 pups. Therefore this finding was considered to be not treatment related.
In the group treated at 0.1%, a large proportion of pups were noted to have observations consistent with a lack of, or poor, maternal care (not fed, not cleaned, cold, scattered, cannibalised). These pups (36% by Day 2 post-partum) were either found dead or were prematurely sacrificed due to poor clinical condition. Observations at necropsy confirmed a lack of maternal care (no milk in stomach, cannibalisation). The majority of those pup deaths occurred in six litters and so this was considered not to be an effect of treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
F1a:
There was a slight reduction in mean pup body weight and body weight gain for both males and females at 0.3%, although mean body weight at birth was similar in all groups. The differences were not statistically significant and in the light of findings of the other generations, was considered fortuitous and unrelated to treatment.
F1b:
Mean male and female pup body weight and body weight gain were generally higher in the treated groups compared to controls. The increases were not dose-dependent, not statistically significant and therefore considered unrelated to treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
There was a statistically significant (p<0.01) increase in the time taken for sexual development of females at 0.3%.
There was also a slight increase in the time taken for sexual development for males at 0.3%, which was not statistically significant and within the HCD. However, in the light of the effects observed in the females, an involvement of the test article cannot be entirely excluded.
No difference in body weight of these females was observed. No effect on fertility or mating performance was observed.
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There was no effect on organ weight (incl. pups selected for special necropsy) in males or females.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1a: Apart from the observations related to poor maternal care (see "mortality"), there was one pup with imperforate anus (with filamentous tail) leading to distension of the stomach and intestines. There was a further pup (from a second litter) with a spinal column/tail defect (the tail was in an abnormal position). These findings were considered not treatment-related due to low incidence.
F1b: There was a slightly higher number of pups at 0.1% observed with no milk in stomach, which was due to high values for two dams both of which were noted with high pup mortality in the F1a generation. Furthermore, a number of pups were noted with tail and anal abnormalities, just as in the F1a generation. The low incidence and distribution of these findings, however, precludes an effect of treatment.
There was no effect in pups selected for special necropsy.
Histopathological findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
PUP DEVELOPMENT
F1a:
There was considered to be no effect of treatment on post-natal development of pups as assessed by pinna detachment, eye opening, static righting reflex, startle response and pupillary light reflex.
F1b:
There was considered to be no effect of treatment on post-natal development of pups as assessed by pinna detachment, eye opening, static righting reflex, startle response and pupillary light reflex. In the groups treated at 0.1 and 0.3% the percentage of pups with pinna detachment was slightly lower than that of the control group and other treated groups. This was due to lower values for up to two litters in each group and was thought not to be an effect of treatment.

F1 GENERATION DEVELOPMENTAL OBSERVATIONS
There was no no effect on startle reflex screening in F1 animals. Time to maximum response amplitude was similar between groups. With respect to the maximum response amplitude, the response was generally lower in females at all dose levels (compared to males), but was probably attributable to body weight differences. There were no effects on learning and memory as examined by the E-maze. Motor activity was unaffected by treatment.
Developmental immunotoxicity:
not examined
SEXUAL DEVELOPMENT
There was evidence of toxicity on pup development at the top dose that was characterised by an increase in the time taken for sexual development of the male (not significant) and female (significant) offspring. No difference in body weight of these females was observed. No effect on fertility or mating performance was observed. Lastly, the results were within the HCD range. Therefore, it was considered that this effect was non-adverse.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Generation:
F1
Effect level:
0.3 other: %
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related adverse effects observed up to and including the highest dose tested.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
0.3 other: %
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related adverse effects observed up to and including the highest dose tested.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
In the control group and at 0.03%, a large proportion
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
In the control group and at 0.03%, a large proportion of pups were noted to have observations consistent with a lack of or poor maternal care (not fed, not cleaned, cold, scattered, cannibalised). These pups were either found dead or were prematurely sacrificed due to poor clinical condition. This was considered not to be an effect of treatment due to the group distribution of this finding; similar proportions of pups died in the control group and at 0.03%, whereas very few pups died in the group treated at 0.3%.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of treatment on male and female pup body weight and body weight gain. Mean values were generally higher than those of the control group, but statistical significance was not attained.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There was no effect on organ weight (incl. pups selected for special necropsy) in males or females.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Apart from the observations related to poor maternal care (i.e, pups with no milk in stomach, see "mortality"), a number of pups at 0.03% were noted with major cranio-facial abnormalities. All pups with this observation were from the same litter. It was considered unrelated to treatment.
There was no effect in pups selected for special necropsy.
Histopathological findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There was considered to be no effect of treatment on post-natal development of pups as assessed by pinna detachment, eye opening, static righting reflex, startle response and pupillary light reflex.
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Generation:
F2
Effect level:
0.3 other: %
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related adverse effect observed up to and including the highest dose tested.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F2
Effect level:
0.3 other: %
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment-related adverse effect observed up to and including the highest dose tested.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
In the present two-generation reproductive toxicity study in the rat, which was compliant with OECD 416 and performed under GLP conditions, rats were treated with 0.03, 0.1 and 0.3% test substance in drinking water. In summary, there was no effect of treatment at any dose level neither on reproduction nor systemic toxicity of the parents or offspring. The reproductive/developmental and the systemic NOAEL were therefore considered to be ≥0.3 %;, corresponding to roughly ≥ 300 mg/kg bw/day.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The whole data base is conclusive and of high quality.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The assessment is based on the data currently available. New studies, based on the category review and the final decisions issued for some of the category substances, which are also relevant for this assessment, are currently being conducted. The hazard assessment with respect to toxicity to reproduction will be updated once all ongoing studies have been finalised.

One study on reproductive toxicity is available for AES (C12-14) Na (CAS 68891-38-3). In a two-generation reproduction study with AES (C12-14) Na (CAS 68891-38-3, 27% a.i.)(BASF, 1999) Sprague Dawley rats were dosed via drinking water at 0, 0.03, 0.1 and 0.3%, which corresponded to daily doses of ca. 0, 30, 100 and 300 mg/kg bw/day. There were some changes indicative of parental toxicity in the group treated with 0.3% of the test substance. Slight but significantly reduced straight line velocity (VSL) of the sperm was without any significant effects on averaged path velocity (VAP) or total motility. Moreover, in the available subchronic and chronic toxicity studies on various AES the primary sex organs of the males and females did not show evidence for treatment-related adverse effects. The observed reduced triglyceride levels (female) and increased percentage neutrophil counts (males) were slight and within the range of the historical control data. The male F0 generation showed a small but significant reduction in body weight-liver weight ratios, but the corresponding brain related liver weights and the absolute liver weights developed not in a dose dependent way. For the F1 generation where similar results were reported, no dose-response relationship was detected either. No influence on liver weight development was seen in the F2 generation. None of the groups revealed any histopathological or clinical-chemical findings, which could be attributed to hepatotoxicity. This led to the conclusion that this untypical liver weight reduction was of no toxicological relevance, additionally underlined by the absence of such effects in the studies for subchronic toxicity mentioned above. There was evidence of toxicity on pup development at this dose level that was characterised by an increase in the time taken for sexual development of the male (not significant) and female (significant) offspring. This was investigated in more detail in the developmental toxicity study up to 1500 mg/kg bw/day (see "Developmental toxicity") and no effects were noted there. Considering all these facts, the subchronic NOAEL for systemic toxicity can be set to greater than 300 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

The assessment is based on the data currently available. New studies, based on the category review and the final decisions issued for some of the category substances, which are also relevant for this assessment, are currently being conducted. The hazard assessment with respect to developmental toxicity will be updated once all ongoing studies have been finalised.

OECD 414, rat, developmental toxicity, oral: not teratogenic

NOAELdev = 1000 mg/kg bw/day; LOAELdev > 1000 mg/kg bw/day

OECD 414, rabbit, developmental toxicity, oral: not teratogenic

NOAELdev = 300 mg/kg bw/day; LOAELdev > 300 mg/kg bw/day

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 June - 15 July 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 1981
Deviations:
yes
Remarks:
Administration comprised only period of organogenesis.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
CD
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Wiga Sulzfeld, Germany
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: approx. 204 g
- Number of animals: 96, primiparous time-mated females
- Housing: Individually in Makrolon Type M3 cage (Ebeco, 44579 Castrop-Rauxel, Germany) with standard softwood bedding (ARWI-Center, 45307 Essen, Germany)
- Diet: Pelleted Altromin Maintenance Diet 1324, ad libitum
- Water: Community tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 23
- Humidity (%): 42 - 66
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 8 June 1993 To: 15 July 1993
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Aqua dest.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared daily by dissolving appropriate amounts of the test material in water yielding a final concentration of 1, 3, and 10% corresponding to 100, 300 and 1000 mg/kg bw/day, respectively.

VEHICLE:
- Concentration in vehicle: 1, 3 and 10%
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The formulation was analysed by "Henkel TTA-Zentrale Analytik" in the time from 21 - 30 June 1993 (study number TTA 93-6431). The concentrations of the test substance in aqua dest. based upon the results of the determination of the dry sediment confirmed the active content of 70.1% of the test substance.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
The females were mated at the supplier with an accurate day of mating. They were received at the testing facility on day 0 of gestation.
The mated animals (number) were delivered:
June 08, 1993 (24)*
June 09, 1993 (24)
June 23, 1993 (24)
June 24, 1993 (18)
June 25, 1993 (16)**
*: on delivery 3 animals dead, 1 animal died one day later
**: 15 females used in study
Duration of treatment / exposure:
Day 6-15 (incl.), post coitum
Frequency of treatment:
daily, 7 days/week
Duration of test:
Until Day 20 post coitum
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
corrected for a.i. content of test material
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
corrected for a.i. content of test material
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
corrected for a.i. content of test material
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose selection is based on the results of an earlier toxicoligical investigation. Rats (10 animals/dose/sex) were orally exposed to 100, 500 and 1000 mg/kg bw/day for 28 days (BASF, 1985, TBD870334). Adverse effects (lesions in the mucosa of the forestomach) were observed at 500 and 1000 mg/kg bw/day. In addition, alterations in hematology and clinical chemistry parameters were observed in the mid- and high-dose group animals when compared to the controls. Therefore, 100, 300 and 1000 mg/kg bw/day were selected as the dose levels for the pre-natal developmental toxicity study since the females in this study were dosed on only 10 consecutive days (Day 6 - 15, inclsive, post coitum).
Maternal examinations:
CAGE SIDE OBSERVATIONS AND MORTALITY: Yes
- Time schedule: twice daily
- Cage side observations included: mortality, signs of reaction to treatment and symptoms of illness

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 16 and 20 post coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 20 post coitum by an overdose of ether
- Organs examined: all maternal organs
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes

Examinations included: Gravid uterus weight, number of corpora lutea, implantations and early/late resorptions

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
Blood sampling:
Not performed.
Fetal examinations:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: No
Statistics:
If the variables could be assumed to follow a normal distribution, the Dunnett-Test, based on a pooled variance, was applied for the comparison between the treated groups and the control group.
The Steel-Test was applied when the data could not be assumed to follow a normal distribution.
Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomised without loss of information (Bonferroni-Holm-corrected).
Indices:
No information reported.
Historical control data:
No data reported.
Clinical signs:
no effects observed
Description (incidence and severity):
No test substance-related symptoms were observed in any animal at any dose level.
Mortality:
no mortality observed
Description (incidence):
No deaths occured in any dams of the control and treatment groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gains were essentially similar in all groups and comparable with the controls.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not specified
Description (incidence and severity):
The following parameter relevant to ED were examined:
- gravid uterus weight, placenta weight
- number of corpora lutea / implantations
- number of early and late resorptions
- pre-implantation loss, post-implantation loss
- number of live and dead fetuses
- sex ratio
- fetal weights
- fetal abnormalities (skeletal, visceral and external)
For details, please refer to the respective result section.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No effect on gravid uterus weight and placental weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic changes were noted in the dams of the control and treatment groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
In the mid-dose group (300 mg/kg bw/day), the mean value of the pre-implantation loss was decreased (level 5%). The finding was considered incidental and in the normal range.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Remarks:
corrected for a.i. content of test material
Basis for effect level:
other: No treatment-related adverse effects observed up to and including the highest dose tested.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
With the exception of the male group 2 (slight mean decrease) the weights of live fetuses exhibited no significant differences on a litter and individual basis. Mean weight between the control group and the treatment group was comparable.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
In the high-dose group (1000 mg/kg bw/day), the mean value of the total males was increased (level 5%) and that of the total females decreased (level 5%). These findings were considered to be incidental and in the normal range.
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
In the high dose group (1000 mg/kg bw/day), the mean value of total males was increased (5%) and the corresponding value for total females decreased. This finding was considered incidental and within the normal range.
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No macroscopical findings were noted at external examination of fetuses which were considered to be an effect of the treatment with the test article. In group 4, there was one foetus with paleness.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Retardations were noted in group 3 and 4. The finding "two sternebrae, non ossified" (p<0.05) is not a negative effect of the substance because a decrease indicates a more developed embryonic stage than in the other fetuses. It was considered incidental. Incidences were 14-12-18-4 for the control, 100, 300 and 1000 mg/kg bw/day group, respectively.
Furthermore, the retardation effect of the "hyoid, incomplete ossified" (p<0.01) is also incidental and not dose-related. Incidences were 1-3-16-3 in the control, 100, 300 and 1000 mg/kg bw/day group, respectively. Only singular litters were affected. The incidental character of this variation is emphasised by the fact the values were within the normal range of variation of this strain.
No variations were observed.
No malformations were observed in treated groups.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects.
The incidences of the most common findings are listed below:
Hydronephrosis: 24/150, 31/156, 21/173 and 34/161 affected foetuses in Group 1, 2, 3 and 4, respectively.
Waved ureter: 11/150, 7/156, 8/173 and 19/161 affected foetuses in Group 1, 2, 3 and 4, respectively.
Dilatation of ureter: 9/150, 8/156, 4/173 and 18/161 affected foetuses in Group 1, 2, 3 and 4, respectively.
There was no dose-response and therefore, no relationship to treatment was inferred.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Remarks:
corrected for a.i. content of test material
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
In the present prenatal developmental toxicity study compliant with OECD 414 (with deviations) and GLP, rats were dosed orally at doses 100, 300 and 1000 mg/kg bw/day during gestation day 6-15. The results of this study showed that repeated oral administration of the test substance to pregnant rats caused no symptoms of cumulative toxicity and did not reveal any embryotoxic or teratogenic potential up to a dose level of 1000 mg/kg bw/day.
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13.
Reason / purpose for cross-reference:
read-across source
Species:
rabbit
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No relevant treatment-related effects observed.
Remarks on result:
other: Source, key, 68585-34-2, 1972
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 165 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: No relevant treatment-related effects observed.
Remarks on result:
other: Source, key, 68585-34-2, 1972
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No relevant treatment-related effects observed.
Remarks on result:
other: Source, key, 68585-34-2, 1972
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
>= 165 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: No relevant treatment-related effects observed.
Remarks on result:
other: Source, key, 68585-34-2, 1972
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
The NOAEL for the test substance was 300 mg/kg bw/day on the basis that the test substance did not produce a significant increase in the number of abnormal fetuses at any dose level. There were no significant test substance-related differences in the numbers of corpora lutea, implantation, resorptions or dead fetuses. Under the conditions of this study, the test substance, at the levels tested, was neither embryotoxic nor teratogenic.
Executive summary:

The developmental toxicity of the target substance is estimated based on an adequate and reliable prenatal developmental toxicity key study of a structural analogue source substance. The study used rabbits as test animals and revealed that the test substance did not produce a significant increase in the number of abnormal fetuses at any dose level. There were no significant test substance-related differences in the numbers of corpora lutea, implantation, resorptions or dead fetuses. Hence, under the conditions of the study, the test substance was neither embryotoxic nor teratogenic and the developmental NOAEL was determined to be > 300 mg/kg bw/day in rabbits. The results of the key study are further supported by additional (supporting) studies on varous analogue source substances. No relevant hazard with regard to developmental toxicity is therefore identified for the target substance as well. As explained in the category justification, the differences in molecular structure between the target and the source substances are unlikely to lead to differences with respect to developmental toxicity or teratogenicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The whole data base is conclusive and of high quality.
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

The assessment is based on the data currently available. New studies, based on the category review and the final decisions issued for some of the category substances, which are also relevant for this assessment, are currently being conducted. The hazard assessment with respect to developmental toxicity will be updated once all ongoing studies have been finalised.

Data on developmental toxicity are available for various members of the AES category The AES reported within the category show similar structural, physico-chemical, environmental and toxicological properties. The approach of grouping different AES for the evaluation of their effects on human health and the environment was also made by the Danish EPA (2001) and HERA (2003), supporting the read-across approach between structurally related AES.

The purpose of the key study (BASF, 1994f) was to assess the effects of orally administered AES (C12-14) Na (CAS 68891-38-3) on embryonic and fetal development in pregnant CD rats. The study followed OECD Guideline 414 and complied with the OECD principles of GLP. In this study, AES (C12-14) Na (CAS 68891-38-3) was administered orally by gavage at dose levels of 0, 100, 300, and 1000 mg/kg bw/day once daily on Day 6-15 of gestation. In summary, the results of the study showed that repeated oral administration of AES (C12-14; 2EO) Na (CAS 68891-38-3) to pregnant rats did not cause symptoms of cumulative toxicity up to a dose level of 1000 mg/kg bw/day. There were no treatment-related fetal abnormalities at necropsy and no treatment-related effects in the reproduction data. Thus, based on the available information, the NOAEL for teratogenicity and developmental toxicity is assessed to be greater than 1000 mg/kg bw/day.

 

Pregnant albino rabbits, artificially inseminated, were administered AES (C10-16; 3 EO) Na (CAS 68585-34-2) by gavage at levels of 50, 100, or 300 mg/kg bw/day on Days 2-16 of gestation (P&G, 1972).No maternal deaths were attributable to the test substance and there were no significant differences in the number of corpora lutea, resorptions, implantations or live fetuses. The number of dead fetuses, 19 in the high level treatment group (300 mg/kg bw/day) and 17 in the control group, were higher than the remaining groups. All but four of these deaths occurred in just three litters. This lack of dose response indicated that something other than the test substance caused the deaths of these fetuses. In addition, no statistical differences were seen in the fetal weights of survivors or with regard to the number of fetuses with skeletal defects. The examination of fetuses from this study for soft-tissue abnormalities revealed only one instance of significant difference occurring in one litter of the low level treatment group. The lack of a dose response relationship between the levels of the test substance and the incidences of stressed bladder (five) indicate an isolated incidence of spontaneous malformations which have been seen in previous teratology studies. The NOAEL for the test substance was 300 mg/kg bw/day on the basis that the test substance did not produce any significant increases in the number of abnormal fetuses at any dose level. There were no significant test substance-related differences in the numbers of corpora lutea, implantation, resorptions or dead fetuses. Under the conditions of this study, the test substance, at the levels tested, was neither embryotoxic nor teratogenic.

 

References:

Danish EPA - Environmental and Health Assessment of Substances in Household Detergents and Cosmetic Detergent Products (2001). Environmental Project No. 615, pp. 24-28

HERA (2003). Human & Environmental Risk Assessment on ingredients of European household cleaning products Alcohol Ethoxysulphates, Human Health Risk Assessment Draft, 2003. http: //www. heraproject. com.

Justification for classification or non-classification

The available data on toxicity to reproduction do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.

Additional information