Alcohols, C12-14, ethoxylated, sulfates, sodium salts

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C12-14, ethoxylated, sulfates, sodium salts

EC Number:

500-234-8

EC Name:

Alcohols, C12-14, ethoxylated, sulfates, sodium salts

Cas Number:

68891-38-3

Molecular formula:

not applicable, UVCB

IUPAC Name:

Alcohols, C12-14(even numbered), ethoxylated < 2.5 EO, sulfates, sodium salts

Details on test material:

- Name of test material (as cited in study report): Trade name
- EC 500-234-8
- CAS 68891-38-3
- Ethoxylation degree: No data
- Physical state: Aqueous solution
- Analytical purity: 27% a.i.
- Lot/batch No.: 103/96
- Storage Condition of test material: At RT in the dark

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

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.

Doses / concentrations 2

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.

Doses / concentrations 3

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.

Doses / concentrations 4

Dose / conc.:

0.015 other: % in water

Remarks:

Females of group 2 during lactation

Doses / concentrations 5

Dose / conc.:

0.05 other: % in water

Remarks:

Females of group 3 during lactation

Doses / concentrations 6

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.

Examinations

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)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

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.

Effect levels (P0)

1

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.

2

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.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Details on results (P1)

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.

Effect levels (P1)

1

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.

2

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.

Target system / organ toxicity (P1)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

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 (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

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.

Effect levels (F1)

1

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

2

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

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 (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

1

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

2

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

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

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.