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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
before January 1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
- Effects upon reproductive function (including two-generation reproductive toxicity)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
before January 1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Principles of method if other than guideline:
Feeding study on both reproductive function and intra-uterine development in the rat, 3 dose groups were fed diets containing 1000, 3000 and 10000 ppm test material.
For assessment of reproductive function: 25 male and 25 female rats of F0 and F1 generation/group were fed diets continuously for 60 days prior to mating and throughout three successive pregnancies, interim kill after 60 days of treatment on 5 males and 5 females, terminal sacrifice of 20 male and 20 female animals following the 3rd mating on Day 13 and in Day 21 of gestation.
Endpoints investigated: clinical signs, body weight/body weight gain, compound intake, haematology, reproductive parameters, litter observations, gross necropsy, organ weights, histology.
GLP compliance:
no
Limit test:
no
Justification for study design:
The study was planned and conducted before 1978, therefore the test design is different to nowadays standard. However, the test design does include e.g. a pre-mating treatment period (60 days), mating of the F1 animals to produce an F2 generation and a 21-day rearing period of the offspring and is therefore considered acceptable for the assessment.
Specific details on test material used for the study:
Information from study report:
- Name of test material: Hostapur SAS 60
- Appearance/Further information: aqueous slurry (60 % w/w active ingredient); Hostapur (SAS 60) is
equal to 60 % Hostapur SAS 93 in water
- Batch number: batch received on 3 September 1974 (lot number NR T 2/112)
- Purity (of Hostapur SAS 93): 91-95 % active ingredient
- Solubility and stability of the test substance in the diet: No data
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Sprague Dawley CD strain
- Source: Charles River U.K. Ltd., Margate, Kent, England
- Weight at study initiation (mean): F0 males 78-80 g, F0 females 87-89 g
- Housing: high density polypropylene and stainless steel cages (Type RC1 and RM2 from North Kent Plastics Ltd., Dartford, Kent)
- Diet and water: ad libitum
- Acclimation period: "short acclimation period" is stated in report, but not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
No further data
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Remarks:
substance given in complete rodent diet
Details on exposure:
For converting substance concentrations in feed into daily doses the factor of 0.09 was used (default factor for subchronic studies, i.e. two-generation study, for rats; Guidance on selected default values to be used by the EFSA Scientific Committee, Scientific Panels and Units in the absence of actual measured data, EFSA Journal 2012;10(3):2579)
Details on mating procedure:
Following the pre-mating period, twenty females from each group were paired on a one to one basis with males from the same treatment level avoiding sibling matings wherever possible. Each morning following pairing, vaginal smears were taken from all females and examined for the presence of spermatozoa. The day on which a sperm positive smear was found was designated Day 1 of gestation. Once mating had occurred, the males and females were separated.
Females failing to mate within five days were placed with another male; this was repeated on two further occasions thereby allowing each female a maximum of 20 days in which to mate.
Approximately 20 days after weaning of the first (A) litters the females were re-mated with different males to produce the second (B) litters. This was repeated a second time to produce the third (C) litters.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Treatment was given continuously to both sexes of F0 and F1 generations for 60 days prior to mating and throughout three successive pregnancies.
Frequency of treatment:
not applicable, diet was freely available to rats
Details on study schedule:
The study on toxicity to reproduction investigates both effects upon reproductive function and developmental toxicity.The complete study extended over three pregnancies derived from each of two generations (F0, F1).
Dose / conc.:
90 mg/kg bw/day
Remarks:
Corresponding dose for group receiving diet with 1000 ppm test substance
Dose / conc.:
270 mg/kg bw/day
Remarks:
Corresponding dose for group receiving diet with 3000 ppm test substance
Dose / conc.:
900 mg/kg bw/day
Remarks:
Corresponding dose for group receiving diet with 10000 ppm test substance
No. of animals per sex per dose:
F0- and F1-generation: 20; Within each group 25 male and 25 female animals were initially used, but interim investigations and sacrifice was carried out after 60 days of treatment on 5 males and 5 females in each group, lowering the number of animals per sex and dose to 20.
Control animals:
yes, plain diet
Details on study design:
In both the F0 and F1 generations, haematological investigations were carried out after 60 days of treatment on five males and five females in each group prior to subsequent macroscopic and histopathological examination. The remaining animals were paired, within groups, on a one to one basis on three consecutive occasions. After the first two matings (F1A, F1B, F2A, F2B) the females were allowed to litter naturally and rear their young to weaning. Following the third mating the dams were killed on Day 13 and 21 of gestation, to permit examination of their uterine contents (see chapter 7.8.2 "Developmental toxicity").
Within each group 25 male and 25 female weanlings were selected at random from amongst those constituting the F1b generation (2nd litter). After a maturation period of 60 days, these animals were mated to produce the F2 generation, using the same procedures and examinations as were applied to the F0 generation.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined daily for evidence of adverse reaction to treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed weekly throughout the study.

HAEMATOLOGY: Yes
In both the F0 and F1 generations, haematological investigations were carried out after 60 days of treatment on five males and five females in each group. The following parameters were investigated: Erythrocyte Count (RBC), Leucocyte Count (WBC) - total, Haemoglobin Concentration, Packed Cell Volume (PCV), Leucocyte Count (WBC) - differential.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
During the pre-mating period, the food intake per cage of five rats was recorded and the mean intake per rat calculated.
Oestrous cyclicity (parental animals):
From Days 55 to 59 of treatment, daily vaginal smears were taken from all females to determine the presence of oestrus.
Sperm parameters (parental animals):
The length of time that elapsed between pairing and detection of mating was recorded for each mating. This time was termed the pre-coital interval.
Litter observations:
Within 24 hours of birth, the offspring were counted, weighed and examined for external abnormalities. Any dead or moribund pups were noted. Daily records were made of mortalities and litter size. Wherever possible, any pups which died were examined externally and internally in an attempt to determine the cause of death. The offspring were weighed on Days 4 and 21 post partum. The speed of physical development of the offspring was assessed in terms of pinna unfolding, hair growth, tooth eruption and eye opening.
Examinations at weaning were auditory function (assessed using a startle response to a sudden sharp noise) and visual function (assessed by examination of the pupil closure response to a bright point source of light).

For the first two matings in each generation (i.e. F1A and F1B or F2A and F2B) the females were allowed to litter naturally in order to provide the following information: gestation length, litter size at birth, litter weight at birth, viability and normality of offspring, sex of offspring at Day 4 post partum.
Postmortem examinations (parental animals):
Interim kill: On Day 60 following blood sampling, five male and five female rats from each group were killed, a gross macroscopic examination was performed and the following organs removed and weighed: Heart, Kidneys, Liver, Ovaries, Testes. Following weighing, representative samples of these organs, together with those listed below, were conserved prior to histological processing and evaluation: Adrenals, Lung, Pancreas, Small intestine, Spleen, Stomach, Thymus, Urinary bladder.

GROSS NECROPSY
All rats that died were subjected to a thorough macroscopic examination to determine, where possible, the cause of death. At, or shortly after Day 13 or Day 21 of the third pregnancy all parent animals were killed by carbon dioxide asphyxiation and a thorough macroscopic examination carried out.

HISTOPATHOLOGY / ORGAN WEIGHTS
Five males and five females from each group were randomly selected for histopathological evaluation.
The following organs were removed and weighed from five male and five female rats from eachgroup: Heart, Kidneys, Liver, Ovaries/testes. Following weighing, representative samples of these organs, together with those listed below, were conserved prior to histological processing and evaluation: Adrenals, Lung, Pancreas, Small intestine, Spleen, Stomach, Thymus, Urinary bladder.
Postmortem examinations (offspring):
All offspring, except those selected to form the F1 generation, were killed after Day 21 post partum by carbon dioxide asphyxiation and examined for external and gross internal abnormalities. The sex of each offspring was recorded.
Statistics:
The significance of inter-group differences was examined using the Mann-Whitney non-parametric U test in the case of bodyweights and the chi-squared test incorporating Yates' correction in the analysis of pre- and post-implantation losses and viability. Statistical analysis of offspring bodyweight gains was carried out on bodyweight.increments calculated from litter mean offspring weights.
Reproductive indices:
The following indices were calculated: Mean litter size, Mean litter weight, Live birth index
Offspring viability indices:
The following indices were calculated: Mean offspring weight, Mean offspring weight on Day 4 post partum, Viability index
Interim investigations: In both generations prior to mating, food intake, haematological parameters, absolute and relative organ weights and histopathological evaluation of tissues showed no adverse treatment-related effects.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no test substance related mortalities. The general condition of F0 males remained similar to that of the controls throughout the F0 generation. F0 females from treated groups were comparable with control females prior to pairing, but during the reproductive phases females in high and medium dose group (10000 ppm and 3000 ppm, continous treatment) showed a reduction in grooming performance, and an inferior general condition compared with the other groups.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In the F0 generation, slight depression of bodyweight gain was observed in males treated continuously with the test substance at 10,000 ppm. A similar reduction was observed in F0 females treated prior to mating, the effect at 10000 ppm achieving statistical significance (p < 0.05). During the three subsequent pregnancies, some fluctuation in bodyweight gain was recorded in treated females, but no significant inter-group variation was observed. In the F1-generation, a significant depression of bodyweight gain (p < 0.05) was observed in males treated continuously with 3000 or 10000 ppm; those treated with 1000 ppm were only marginally affected. Because of a depressed rate of bodyweight gain during lactation, the mean bodyweight of female offspring from the group treated continously with 10000 ppm was significantly depressed at weaning (p < 0.05). Subsequently however, the rate of bodyweight gain in this group was comparable with that of the controls.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Group mean food consumption of both males and females during the pre-mating period was similar for all groups.

HAEMATOLOGICAL INVESTIGATIONS:
There were no treatment-related inter-group variations in haematological parameters in either male or female animals.

REPRODUCTIVE FUNCTION:
In both generations, oestrous cycles, mating performance and conception rates were unaffected by treatment.

GROSS PATHOLOGY (PARENTAL ANIMALS)/HISTOPATHOLOGY (PARENTAL ANIMALS)
Interim kill: No treatment related findings were reported.
Macroscopic examination, absolute and relative organ weights and histopathological evaluation of F0 and F1 parent animals showed no adverse treatment-related effects.
Dose descriptor:
NOAEL
Remarks:
General Toxicity
Effect level:
ca. 270 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Slight depression of somatic growth in next higher dose group
Dose descriptor:
NOAEL
Remarks:
Fertility
Effect level:
ca. 900 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No dose related effect on fertility consistent over two generations reported
see Details on results P0
Dose descriptor:
NOAEL
Remarks:
General Toxicity
Effect level:
ca. 270 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Slight depression of somatic growth in next higher dose group
Dose descriptor:
NOAEL
Remarks:
Fertility
Effect level:
ca. 900 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No dose related effect on fertility consistent over two generations reported
VIABILITY (OFFSPRING)
The viability index was significantly depressed (p < 0.05) in the F1A litters of females receiving 3000 ppm or 10000 ppm continuously, and in the F2B litters of the females receiving 3000 ppm continuously. In all other groups, the viability index was comparable with that of the control group.

CLINICAL SIGNS (OFFSPRING)
A marginal delay in physical development of F2B offspring was recorded in all treated groups.

BODY WEIGHT (OFFSPRING)
The bodyweight of offspring at Day 1 post partum, showed no significant inter-group variations. However, the bodyweight gain of offspring from females receiving 10000 ppm continuously was depressed in the F1A, F1B, F2A and F2B litters, achieving statistical significance (p < 0.05) in all but the F2A litters. All other treated offspring gained weight at a similar rate to the controls.

GROSS PATHOLOGY (OFFSPRING)
No indication of any treatment-related findings.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 270 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Slight depression of somatic growth in next higher dose group
See Details on results (F1)
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
ca. 270 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Slight depression of somatic growth in next higher dose group
Reproductive effects observed:
no
Executive summary:

The influence of the test material upon reproductive function and fertility was assessed over two generations in rats of the Charles River CD strain. For this purpose, the substance was administered in the diet to both the F0 and F1 generations at levels of 1000, 3000 or 10000 ppm. Treatment was given either continuously to both sexes for 60 days prior to mating and throughout three successive pregnancies (F1A, F1B, F1C, F2A, F2B and F2C) or to females only during the organogenesis stage of three successive pregnancies. Animals were randomly selected from the F1B litters to form the second generation.

In both the F0 and F1 generations, haematological investigations were carried out after 60 days of treatment on five males and five females in each group, prior to carbon dioxide asphyxiation and subsequent macroscopic and histopathological examination. The remaining animals were paired, within groups, on a one to one basis on three consecutive occasions. After the first two matings (F1A, F1B, F2A, F2B) the females were allowed to litter naturally and rear their young to weaning. Following the third mating (F1C, F2C) half of the dams in each group were killed on day 13 of gestation, and the remainder were killed on day 21 of gestation, to permit examination of their uterine contents.

After termination of each generation, all parent animals were examined macroscopically. Five males and five females from each of the continuously treated groups, together with five females only from each of the groups treated during organogenesis, were randomly selected for histopathological evaluation.

In both generations prior to mating, food intake, haematological parameters, absolute and relative organ weights and histopathological evaluation of tissues showed no adverse treatment-related effects.

In the F0 generation, slight depression of bodyweight gain was observed in males treated continuously with the substance at 10,000 ppm. A similar reduction was observed in F0 females treated prior to mating. During the three subsequent pregnancies, some fluctuation in bodyweight gain was recorded in treated females, but no significant inter-group variation was observed.

In the F1 generation, a slight depression of bodyweight gain was observed in males treated continuously with 3,000 or 10,000 ppm; those treated with 1,000 ppm were only marginally affected.

Because of a depressed rate of bodyweight gain during lactation, the mean bodyweight of female offspring from the group treated continuously with 10,000 ppm was significantly depressed at weaning. Subsequently however, the rate of bodyweight gain in this group was comparable with that of the controls. With the exception of females receiving 10,000 ppm during organogenesis, in which a significant reduction in bodyweight was recorded in the third pregnancy, no significant inter-group variations in bodyweight gain were observed.

Neither generation, during the first two pregnancies (F1A, F1B, F2A, F2B), showed any treatment-related effects in the number of litters containing at least one viable young, the litter size at birth, or the live birth index.

The viability index was significantly depressed in the F1A litters of females receiving 3000 or 10000 ppm continuously, and in the F2B litters of the females receiving 3000 ppm continuously. These observations are not regarded as compound related adverse effects because no consistent dose response or consistent effect over generations is observed. The observation on the F1A litter is due to a single dam that failed to lactate. In all other groups, the viability index was comparable with that of the control group.

The bodyweight of offspring at Day 1 post partum, showed no significant inter-group variations. However, the bodyweight gain of offspring from females receiving 10,000 ppm was depressed in the F1A, F1B and F2B litters, achieving statistical significance. All other treated offspring gained weight at a similar rate to the controls.

In both generations, the sex ratio at Day 4 post partum and at weaning were unaffected by treatment. A marginal delay in physical development of F2B offspring was recorded in all treated groups with unknown biological significance.

There was no consistent effect on pre-implantation loss, litter parameters or foetal development. Macroscopic examination, absolute and relative organ weights and histopathological evaluation of Fa and FI parent animals showed no adverse treatment-related effects.

It was concluded from these investigations that continuous treatment with the test material at a level of 10,000 ppm gave rise to a slight depression of somatic growth in parent animals and offspring in both generations. There were no indications for an embryotoxic or teratogenic effect related to treatment in any dose groups.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1978
Report Date:
1978

Materials and methods

Principles of method if other than guideline:
Feeding study on both reproductive function and intra-uterine development in the rat, 3 dose groups were fed diets containing 1000, 3000 and 10000 ppm test material.
For assessment of intra-uterine development: 25 females of F0 and F1 generation/group were fed diets during the organogenesis stage (Days 6 to 15 of gestation) of three successive pregnancies, interim kill after 60 days of treatment (5 animals), terminal sacrifice of 20 females following the 3rd mating on Day 13 and Day 21 of gestation.
Endpoints investigated: clinical signs, body weight/body weight gain, compound intake, haematology, gross necropsy, organ weights, histology, evaluation of uterine contents, fetal examinations.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Information from study report:
- Name of test material: Hostapur SAS 60
- Appearance/Further information: aqueous slurry (60 % w/w active ingredient); Hostapur (SAS 60) is equal to 60 % Hostapur SAS 93 in water
- Batch number: batch received on 3 September 1974 (lot number NR T 2/112)
- Purity (of Hostapur SAS 93): 91-95 % active ingredient
- Solubility and stability of the test substance in the diet: No data

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Sprague Dawley CD strain
- Source: Charles River U.K. Ltd., Margate, Kent, England
- Weight at study initiation (mean): F0 males 78-80 g, F0 females 87-89 g
- Housing: high density polypropylene and stainless steel cages (Type RC1 and RM2 from North Kent Plastics Ltd., Dartford, Kent)
- Diet and water: ad libitum
- Acclimation period: "short acclimation period" is stated in report, but not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
No further data

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Remarks:
(substance given in complete rodent diet)
Details on exposure:
For converting substance concentrations in feed into daily doses the factor of 0.12 was used (default factor for subacute studies for rats, i.e. developmental toxicity study; Guidance on selected default values to be used by the EFSA Scientific Committee, Scientific Panels and Units in the absence of actual measured data, EFSA Journal 2012;10(3):2579).
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Following the pre-mating period, twenty females from each group were paired on a one to one basis with males avoiding sibling matings wherever possible. Each morning following pairing, vaginal smears were taken from all females and examined for the presence of spermatozoa. The day on which a sperm positive smear was found was designated Day 1 of gestation. Once mating had occurred, the males and females were separated.
Females failing to mate within five days were placed with another male; this was repeated on two further occasions thereby allowing each female a maximum of 20 days in which to mate.
Approximately 20 days after weaning of the first (A) litters the females were re-mated with different males to produce the second (B) litters. This was repeated a second time to produce the third (C) litters.
Duration of treatment / exposure:
Treatment was given to females during the organogenesis stage (Days 6 to 15 of gestation) of three successive pregnancies.
Frequency of treatment:
not applicable, diet was freely available to rats.
Duration of test:
The study on toxicity to reproduction investigates both effects upon reproductive function and developmental toxicity. Therefore no data is given for the duration of the developmental part of the test. The complete study extended over three pregnancies derived from each of two generations (F0, F1).
Doses / concentrationsopen allclose all
Dose / conc.:
120 mg/kg bw/day
Remarks:
Corresponding dose for group receiving diet with 1000 ppm test substance
Dose / conc.:
360 mg/kg bw/day
Remarks:
Corresponding dose for group receiving diet with 3000 ppm test substance
Dose / conc.:
1 200 mg/kg bw/day
Remarks:
Corresponding dose for group receiving diet with 10000 ppm test substance
No. of animals per sex per dose:
F0- and F1-generation: 20; Within each group 25 female animals were initially used, but interim investigations and sacrifice was carried out after 60 days on 5 females in each group, lowering the number of animals per sex and dose to 20.
Control animals:
yes, plain diet
Details on study design:
In both the F0 and F1 generations, haematological investigations were carried out after 60 days on five females in each group prior to subsequent macroscopic and histopathological examination. The remaining animals were paired, within groups, on a one to one basis on three consecutive occasions. After the first two matings (F1A, F1B, F2A, F2B) the females were allowed to litter naturally and rear their young to weaning. Following the third mating (F1C, F2C) half of the dams in each group were killed on Day 13 of gestation, and the remainder were killed on Day 21 of gestation, to permit examination of their uterine contents.
Within each group 25 male and 25 female weanlings were selected at random from amongst those constituting the F1b generation (2nd litter). After a maturation period of 60 days, these animals were mated to produce the F2 generation, using the same procedures and examinations as were applied to the F0 generation.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined daily for evidence of adverse reaction to treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on Days 1 and 60 before mating and thereafter at weekly intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
During the pre-mating period, the food intake per cage of five rats was recorded and the mean intake per rat calculated.

POST-MORTEM EXAMINATIONS: Yes
All rats that died were subjected to a thorough macroscopic examination to determine, where possible, the cause of death. Following the third mating (F1C, F2C) half of the dams in each group were killed on Day 13 of gestation, and the remainder were killed on Day 21 of gestation, to permit examination of their uterine contents.

HISTOPATHOLOGY / ORGAN WEIGHTS
Five females from each group were randomly selected for histopathological evaluation.
The following organs were removed and weighed from five male and five female rats from eachgroup: Heart, Kidneys, Liver, Ovaries/testes. Following weighing, representative samples of these organs, together with those listed below, were conserved prior to histological processing and evaluation: Adrenals, Lung, Pancreas, Small intestine, Spleen, Stomach, Thymus, Urinary bladder.
Ovaries and uterine content:
After the third mating (F1C or Fl2), each group was randomly divided into two sub-groups of approximately equal size. One sub-group from each level was killed on Day 13 post coitum whilst the other was killed on Day 21 post coitum. The following information was recorded for each female:
Day 13 - number of corpora lutea, number of implantation sites, number of resorption sites, number and viability of foetuses.
Day 21 - number of corpora lutea, number of implantation sites, number of resorption sites, number, viability and normality of foetuses, weight and sex of viable foetuses.
Fetal examinations:
Following gross examination, one-third of each litter was placed in Bouin's fixative for subsequent free-hand sectioning, following the technique of Wilson (in Teratology: Principles and Techniques, p 251, Univ. Chicago Press, 1965). The thoracic and abdominal cavities of the remainder were dissected and examined under low-power magnification. Following examination and evisceration, the foetuses were placed in 95% industrial methylated spirit prior to processing, which utilized a modification of the Dawson staining technique (Dawson, A.B., Stain Technol., 1, 123, 1926), and subsequent skeletal examination.
Statistics:
The significance of inter-group differences was examined using the Mann-Whitney non-parametric U test in the case of bodyweights and the chi-squared test incorporating Yates' correction in the analysis of pre- and post-implantation losses and viability. Statistical analysis of offspring bodyweight gains was carried out on bodyweight.increments calculated from litter mean offspring weights.
Indices:
The following indices were calculated for dams killed on Day 13 or Day 21 of gestation: Pre-implantation loss, Post-implantation loss

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
For females receiving 10000 ppm during organogenesis, a significant reduction (p < 0.05) in bodyweight was recorded in the third pregnancy.

Macroscopic examination, absolute and relative organ weights and histopathological evaluation of F0 and F1 parent animals showed no adverse treatment-related effects.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
ca. 360 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
body weight and weight gain

Results (fetuses)

Details on embryotoxic / teratogenic effects:
In the F0 generation, a significant prolongation (p < 0.05) of gestation occurred in the second pregnancy (F1B) of females treated with 10000 ppm during organogenesis. However, the range of gestation lengths was simllar to that of the control group in the F1A litter and to those of the F1 generation. No other alterations in duration of gestation were observed.

In the F0 animals killed on Day 13 post coitum (F1C) there were some inter-group variations in the extent of pre-implantation loss, but only in females receiving 1000 ppm during organogenesis was there any significant increase (p < 0.05). In the F1 animals killed on Day 13 of gestation (F2C) there was an apparent increase in pre-implantation loss in treated females, but this was due to an unusually low control value rather than to an test substance related adverse effect.

In both generations, although there were some minor inter-group variations in litter parameters and foetal development of females killed on Day 21 post coitum, the differences did not achieve statistical signiflcance .

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
ca. 1 200 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
other: No teratogenic effect observed at any dose tested.

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Executive summary:

The influence of the test material upon reproductive function and fertility was assessed over two generations in rats of the Charles River CD strain. For this purpose, the substance was administered in the diet to both the F0 and F1 generations at levels of 1000, 3000 or 10000 ppm. Treatment was given either continuously to both sexes for 60 days prior to mating and throughout three successive pregnancies (F1A, F1B, F1C, F2A, F2B and F2C) or to females only during the organogenesis stage of three successive pregnancies. Animals were randomly selected from the F1B litters to form the second generation.

In both the F0 and F1 generations, haematological investigations were carried out after 60 days of treatment on five males and five females in each group, prior to carbon dioxide asphyxiation and subsequent macroscopic and histopathological examination. The remaining animals were paired, within groups, on a one to one basis on three consecutive occasions. After the first two matings (F1A, F1B, F2A, F2B) the females were allowed to litter naturally and rear their young to weaning. Following the third mating (F1C, F2C) half of the dams in each group were killed on day 13 of gestation, and the remainder were killed on day 21 of gestation, to permit examination of their uterine contents.

After termination of each generation, all parent animals were examined macroscopically. Five males and five females from each of the continuously treated groups, together with five females only from each of the groups treated during organogenesis, were randomly selected for histopathological evaluation.

In both generations prior to mating, food intake, haematological parameters, absolute and relative organ weights and histopathological evaluation of tissues showed no adverse treatment-related effects.

In the F0 generation, slight depression of bodyweight gain was observed in males treated continuously with the substance at 10,000 ppm. A similar reduction was observed in F0 females treated prior to mating. During the three subsequent pregnancies, some fluctuation in bodyweight gain was recorded in treated females, but no significant inter-group variation was observed.

In the F1 generation, a slight depression of bodyweight gain was observed in males treated continuously with 3,000 or 10,000 ppm; those treated with 1,000 ppm were only marginally affected.

Because of a depressed rate of bodyweight gain during lactation, the mean bodyweight of female offspring from the group treated continuously with 10,000 ppm was significantly depressed at weaning. Subsequently however, the rate of bodyweight gain in this group was comparable with that of the controls. With the exception of females receiving 10,000 ppm during organogenesis, in which a significant reduction in bodyweight was recorded in the third pregnancy, no significant inter-group variations in bodyweight gain were observed.

Neither generation, during the first two pregnancies (F1A, F1B, F2A, F2B), showed any treatment-related effects in the number of litters containing at least one viable young, the litter size at birth, or the live birth index.

The viability index was significantly depressed in the F1A litters of females receiving 3000 or 10000 ppm continuously, and in the F2B litters of the females receiving 3000 ppm continuously. These observations are not regarded as compound related adverse effects because no consistent dose response or consistent effect over generations is observed. The observation on the F1A litter is due to a single dam that failed to lactate. In all other groups, the viability index was comparable with that of the control group.

The bodyweight of offspring at Day 1 post partum, showed no significant inter-group variations. However, the bodyweight gain of offspring from females receiving 10,000 ppm was depressed in the F1A, F1B and F2B litters, achieving statistical All other treated offspring gained weight at a similar rate to the controls.

In both generations, the sex ratio at Day 4 post partum and at weaning were unaffected by treatment.

A marginal delay in physical development of F2B offspring was recorded in all treated groups with unknown biological significance.

There was no consistent effect on pre-implantation loss, litter parameters or foetal development. Macroscopic examination, absolute and relative organ weights and histopathological evaluation of Fa and FI parent animals showed no adverse treatment-related effects.

It was concluded from these investigations that continuous treatment with the test material at a level of 10,000 ppm gave rise to a slight depression of somatic growth in parent animals and offspring in both generations. There were no indications for an embryotoxic or teratogenic effect related to treatment in any dose groups.