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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 19, 1992 to April 19, 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP conditions.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994
Reference Type:
publication
Title:
Acrylic Acid: Two-generation Reproduction Toxicity Study in Wistar Rats with Continuous Administration in the Drinking Water.
Author:
Hellwig J. et al.
Year:
1997
Bibliographic source:
Food and Chemical Toxicology 35: 859-868

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Acrylic acid
EC Number:
201-177-9
EC Name:
Acrylic acid
Cas Number:
79-10-7
Molecular formula:
C3H4O2
IUPAC Name:
acrylic acid
Details on test material:
- Name of test material (as cited in study report): Acrylic acid
- Analytical purity: ≥98.9%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Wistar rats (Chbb = THOM (SPF))
- Source: Karl THOMAE, Biberach an der Riss, Germany
- Age at study initiation: (P): 35 ± 1 days
- Weight at study initiation: (P) Males: 140.0 (127 - 154) g; Females: 118.8 (106 - 130) g
- Housing: Single in type DK III stainless steel wire mesh cages, with the following exceptions: during mating periods, the males designated for mating were kept individually in Makrolon cages, type M III; for the overnight mating the females were put into the cages of the males. From day 18 of pregnancy until day 14 after birth, the pregnant animals and their litters were also housed in Makrolon type M III cages.
- Diet (ad libitum): Kliba maintenance diet rat/ mouse/hamster GLP 343 meal (KLINGENTALMUEHLE AG, Kaiseraugst, Switzerland)
- Water (ad libitum): Tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: a maximum of 3 weeks.
- Proof of pregnancy: sperm in vaginal smear referred as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content of Acrylic acid in the aqueous solutions was determined by gas chromatography.
Duration of treatment / exposure:
Exposure period: F0: at least 70 days before the mating and afterwards during the gestation and lactation periods.
F1: at least 98 days before the mating and afterwards during the gestation and lactation periods.
Premating exposure period (males): at least 70 days
Premating exposure period (females): at least 70 days
Duration of test: approx. 12 months
Frequency of treatment:
continuously
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 2500 and 5000 ppm (corresponding to approx. 0, 53, 240 and 460 mg/kg bw/day)
Basis:
nominal in water
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS:
All parental animals were checked daily for clinically evident signs of toxicity. Particular attention was given to the nesting, littering and lactation behaviour of the dams, but only special findings were documented.

BODY WEIGHT:
- Parental animals: generally, body weight was determined once weekly until the end of the study, and at the time of necropsy.
- F0 and F1 fertilized females and females with litter: body weight was determined on the day of sperm evidence in the vaginal smear and thereafter on days 7, 14 and 20 of gestation, one day after parturition, and on days 7, 14 and 21 post-parturition.
- Females without positive evidence of sperms: body weight was not determined during the mating interval.
- Females without litter: body weight was not determined during the lactation phase.

FOOD CONSUMPTION:
- F0 and F1 parental animals: food consumption was determined once weekly (over 7 days) during the period prior mating.
- Pregnant females: food consumption was determined for days 0-7, 7-14, 14-20 post coitum (pc).
- Lactating females: food consumption was determined for days 0-4, 4-7, 7-14 post parturition (pp).
- F0 and F1 dams between day 14 and 21 pp: food consumption was not determined for the F0 and F1 dams between day 14 and 21 pp, since during this period the pups started consumption of solid food; therefore there was no point in such a measurement.
- Females during mating period, females without positive evidence of sperms, females without litter: food consumption was not determined respectively during mating period, gestation period or lactation phase.


WATER CONSUMPTION:
- F0 and F1 parental animals: water consumption was determined once weekly (over 3 days) during the period prior mating.
- Pregnant females: water consumption was determined for days 0-1, 6-7, 13-14, 19-20 post coitum (pc).
- Lactating females: water consumption was determined for days 1-2, 3-4, 6-7, 13-14 post parturition (pp).
- F0 and F1 dams from day 20 and 21 pp: water consumption was not determined for the F0 and F1 dams for days 20 - 21, since during this period the pups started consumption of water; therefore there was no point in such a measurement.
- Females during mating period, females without positive evidence of sperms, females without litter: water consumption was not determined respectively during mating period, gestation period or lactation phase.


INTAKE OF TEST SUBSTANCE:
The intake of test substance (IT, in mg/kg bw/day) was calculated according to the following formula:

ITx = WCx * D / BWy

D = dose in ppm
WCx = daily water consumption on day x; in g
BWy = body weight on day y; in g


Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
The pups (F1 and F2 litters) were examined as soon as on their day of birth for the determination of the total number of pups and the number of liveborn and stillborn pups (pups died on day of birth prior the first examination). Thereafter the pups were checked twice daily on workdays (once a day on week ends and public holidays) for mortality (i.e. dead and moribund pups) and the mortality (number and percentage) was determined for the day of birth (i.e. day 0) and for the periods: days 1 - 4, 5 - 7, 8 - 14 and 15 - 21 of lactation. Pups that died accidentally and had to be sacrificed because of maternal death were not considered for calculation. The number of surviving pups was determined for days 0, 4, 7, 14 and 21 of lactation and served for the calculation of the viability index and the lactation index.

The sex of the pups was determined on day 0 and day 21 (measurement of the anogenital distance, which is known to be greater in male pups than in females), and the sex ratio was calculated according to following formula:

- Sex ratio = number of live male or female pups on day 0/21 * 100 / number of live male and female pups on day 0/21

The pups were weighed on days 1, 4, 7, 14 and 21 after birth, and they were examined daily for clinical symptoms or gross morphological abnormalities. The determination of the relative organ weight was based on the pup body weight on day 21 after birth. The bodies of the sacrificed pups were examined for external abnormalities and the organs also were subjected to gross pathology; skeletal staining according to the modified Dawson´s method and/or further processing of the head according to Wilson´s method was done in case of abnormal findings. Stillborn pups as well as pups that died during weaning also were subjected to necropsy.

Development stages / Behavioral tests:
Physical development was assessed by monitoring pinna unfolding, opening of the auditory canal and opening of the eyes. Additional tests
were performed to assess grip reflex, hearing and pupillary reflex as follows :
- Grip reflex: Tested on day 13 after birth by placing front paws onto 3-mm diameter rod. For a positive response, the animal had to grip the bar and pull itself up.
- Hearing test: On day 21 after birth, animals were placed in a soundproof box and exposed to a sound (0 .1 sec, 5000 Hz, about 90 dB); a startle reflex was considered a response to this stimulus.
- Pupillary reflex: On day 2l after birth, pupillary constriction reflex was assessed by shining a penlight on the eye and observing the reaction.

Postmortem examinations (parental animals):
SACRIFICE
Parental animals were killed by decapitation under CO2 anaesthesia and examined macroscopically.

GROSS NECROPSY
Terminal body weights as well as the weights of liver, kidneys, epididymides and testes were recorded .

HISTOPATHOLOGY / ORGAN WEIGHTS
Liver, kidneys and stomach (non-glandular and glandular).
Postmortem examinations (offspring):
SACRIFICE
Pups were killed by CO2 asphyxiation, examined externally, eviscerated and their organs assessed macroscopically.


GROSS NECROPSY
External and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY
Vagina, cervix, uterus, ovaries, oviducts, testes, epididymides, seminal vesicles, coagulation gland, oesophagus and duodenum.
Statistics:
The statistical assessment of the different data obtained within the present study was based on following methods, depending on the parameters considered: Dunnett test, Fisher`s exact test and Wilcoxon test.
Reproductive indices:
Mating and fertility indices were calculated according to following formulas:

- Male mating index (%) = number of males with confirmed mating * 100 / number of males placed with females

- Male fertility index (%) = number of males proving their fertility * 100 / number of males placed with females

Remark:
Males were defined as “with confirmed mating” by the presence of vaginal sperm in the female, or by the production of a litter, or by the presence of fetuses in the uterus.
Males were defined as “proving their fertility” by female giving birth to a litter or having pups or fetuses in the uterus.

Reevaluation of fertility:
If an animal of the F0 or F1 generation parental animals had not produced any offspring after the scheduled mating of F0 parents (to get F1 litter) or after the scheduled mating of F1 parents (to get of F2 litter), those animals treated with the test substance were mated with fertile animals of the control groups. Animals of the control groups which seemed to be infertile were mated with mating partners with proven fertility of the controls.
After fertility had been reevaluated, the animals were sacrificed and subjected to gross-pathological and histopathological assessments. The uteri of the females reevaluated for fertility were examined for live and dead implantations. In the case of an apparently non-pregnant animal or of an empty uterus horn in the case of single-horn pregnancy, the uterus was stained with sodium sulfide and assessed for early implantations. Then the uteri were rinsed carefully under running water. After these examinations were completed, the uteri were transferred to the pathology lab for further fixation and evaluation.

Offspring viability indices:
- Viability index (%) = number of live pups on day 4 after birth * 100 / number of liveborn pups on the day of birth

- Lactation index (%) = number live pups on day 21 after birth * 100 / number of live pups on day 4 after birth

Remark:
Day 4 after birth preceded standardization of the litters.
Day 21 after birth followed standardization of the litters.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

**** F0 GENERATION PARENTAL ANIMALS ****

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- There were no mortalities in any of the F0 generation parental animals in any of the groups.
- No clinical signs which might be attributed to the test substance were detected in male or female F0 generation parental animals. The 3 concentrations administered in the drinking water did not lead to disturbances of the general behavior in any of the F0 parental animals.
 - There were no particular substance-related clinical findings in F0 females during the gestation period for F1 litter. Insufficient nesting activity was observed for several dams of all groups including the controls.
 - No substance-related clinical findings were recorded for the F0 dams during the F1 lactation period.

FOOD CONSUMPTION (PARENTAL ANIMALS)
In general, the food consumption of the males (during the premating period) and of the females (during premating, gestation and lactation periods) of all test groups was not influenced by the test substance administration. It was, however, slightly, but statistically significantly reduced in the high dose males during the first week of the premating period and in the females of 5000 ppm test group during the second week of the lactation period.
The sporadic and only marginal reductions in food consumption of the 5000 ppm rats are probably related to the reduced consumption of aqueous acrylic acid solutions of these animals and thus are likely indirectly associated with the administration of the test substance. All other observable differences between the groups are without biological relevance, because they are not dose-related; this includes the statistically significantly increased food consumption of the low dose females during study weeks 0-1 and 6-7 of the premating period.

BODY WEIGHT (PARENTAL ANIMALS)
In the F0 males statistically significant reductions in mean body weights were seen in the highest dose group (5000 ppm) from week 12 until week 20 of the study period. Body weight changes of the high dose males were statistically significantly diminished only at certain study intervals (weeks 0-1, 6-7, 11-12); if calculated for the total study period (weeks 0-20), body weight gain of the high dose males was about 9% lower than that of the respective controls.
Body weights and weight gains of the substance-treated females were similar to control values during the premating period and during gestation and lactation. Only during the first week of gestation did the high dose dams gain statistically significantly less weight than the corresponding controls.
Finally the impairments in body weight/body weight gain in the high dose F0 males and - to a lesser extent - in the F0 females are assessed as being substance-related effects. All other statistically significant differences in body weights and body weight gains are considered unrelated to the test substance because the values were not influenced in a dose-dependent manner and/or are within the biological range of variation.

WATER CONSUMPTION (PARENTAL ANIMALS)
The water consumption of the high dose male and female animals was clearly reduced. This reduction was statistically significant during the premating period. It was also diminished in the 5000 ppm females during gestation and lactation of F1 litter. In total the 5000 ppm males consumed about 11% and the high dose females about 13% less drinking water (aqueous acrylic acid solutions) then the respective controls during the first 10 study weeks. The marked reduction in the drinking water consumption of the high dose rats was associated with the test substance administration. All other observable differences between the groups in respect to water consumption are without biological relevance, because they are not dose-related; this includes the statistically significantly increased water consumption of the 500 ppm female animals during premating weeks 6-7 and 9-10.

MALE REPRODUCTIVE FUNCTION (PARENTAL ANIMALS)
For all F0 males which were placed with females to generate F1 pups mating was confirmed; thus, the male mating index was 100% in all groups. For nearly all F0 males fertility could be confirmed within the scheduled mating interval; the fertility index varied between 92% and 96% with no treatment related effect. Thus, the fertility of the F0 generation parental males was not adversely influenced by the administration of aqueous acrylic acid solutions.

FEMALE REPRODUCTIVE FUNCTION (PARENTAL ANIMALS)
The female mating index calculated after the mating period for F1 litter was 100% for all groups. The mean duration until sperm was detected (day 0 pc) varied between 1.8 and 3.8 days and was statistically significantly longer for the high dose dams; the high dose value (3.8 days), however, is substantially similar to the mean cohabitation time value of the control group (3.2 days) of the second parental generation (F1 animals) and therefore was not considered treatment-related. Only one or two females in all groups, including the controls, did not become pregnant within the scheduled mating interval. The fertility index varied between 92% and 96% without any dose-response relationship. All females in question except the 2 low dose females proved to be fertile after being mated again with control males. The mean duration of gestation was similar in all groups and the gestation index reached 100% for all groups. The mean number of pups delivered/dam was uninfluenced by the test substance administered. The number of liveborn and stillborn pups was comparable between the groups, and the live birth index was 98% in test groups. Thus, the administration of aqueous acrylic acid solutions did not adversely affect reproduction and delivery data of the FO generation parental females.


GROSS AND HISTOPATHOLOGICAL FINDINGS
- Thickening of the limiting ridge (margo plicatus) of the forestomach in most male and female rats.
- Minimal hyperkeratosis at the limiting ridge of the forestomach in most male and all female rats.
- Edema in the submucosa of the glandular stomach of 2 male and 10 female rats, minimal in all cases.

**** F1 GENERATION PARENTAL ANIMALS *****

MORTALITY / CLINICAL OBSERVATIONS
- One male animal of 2500 ppm test group had to be sacrificed in a moribund state due to a severe skin lesion on the base of the tail. There were no other unscheduled mortalities in any of the test groups.
 
- No clinical signs which might have been attributed to the test substance administered were detected in male or female F1 generation parental animals. The 3 doses administered in drinking water did not lead to disturbances of the general behavior in any of the F1 parental animals. One male animal of 2500 ppm test group developed a severe skin lesion on the base of the tail and was sacrificed in a moribund state. Another male of the same dose group showed unilateral chromodacryorrhea. The clinical findings which occurred in just two intermediate dose males were spontaneous in nature.
 
- No particular clinical findings were noted for F1 dams with positive sperm detection except insufficient or no nesting activity, which was recorded for several dams of all groups (0, 500, 2500 and 5000 ppm) and which occurred without a clear dose-response relationship. One female of the low dose group showed vaginal hemorrhage towards or after the end of the gestation period (days 23 - 26 pc), and was not able to deliver the pups, which were palpable earlier in the abdomen of this dam. After day 26 pc, no pups could be palpated for this dam.
 
- There were no substance-related clinical findings in the F1 dams during the lactation of F2 litters. Only one dam of the low dose group and one dam of the high dose group did not nurse their pups properly; all pups of high dose dam were cannibalized and/or died intercurrently. Furthermore, another low dose dam showed blood in bedding during the first days of the lactation period, and was not able to deliver its litter completely. It delivered only 2 pups which were cannibalized on day 1 pp.

FOOD CONSUMPTION
The mean food consumption of the males and females of 5000 ppm test group was clearly reduced during the premating period, the differences in comparison to the controls being statistically significant at most time intervals. In total, the high dose males consumed about 9% and the females about 8% less food than the respective control animals during the premating phase. Food intake was also statistically significantly diminished in the females of this test group (5000 ppm) during the gestation period (days 7-20 pc) and during the lactation period (days 7-14 pp only). The reduction in food consumption of the 5000 ppm males and females was considered to be related to the administration of the test substance. All other differences in food consumption between the groups are without any biological relevance.
 
WATER CONSUMPTION
In comparison to the respective control values the water consumption of the 2500 and 5000 ppm F1 males and females was distinctly lower during the premating period, the differences being statistically significant at all intervals in the high dose level and in several but not all intervals at the intermediate dose. In total a clear dose-response relationship was observed: high dose males consumed about 18%, intermediate dose males about 9% less water than control males; for high dose females about 27% and for intermediate dose females about 13% less water intake than in the female controls was recorded. Water consumption was also reduced during gestation and lactation periods in these test groups, again more pronounced in the high than in the 2500 ppm group. The water consumption of the animals of the low dose group (500 ppm) reached or even exceeded the relevant control values during premating, gestation and the lactation periods. The distinct reductions in the drinking water consumption in both sexes at 2500 and 5000 ppm are considered treatment-related, whereas the differences in water consumption between the low dose group and the control are considered to be without toxicological relevance.
 
BODY WEIGHT
In the F1 males, statistically significant reductions in mean body weights were seen in the highest dose group (5000 ppm) throughout the total study period (about 87% of the control value at the end of this study interval). Body weight gains of the 5000 ppm males, however, were generally similar to the respective control values. In total, the weight gain of the high dose F1 males was only about 5% lower than the body weight gain of the control males. Body weights of the 5000 ppm females were also statistically significantly reduced during the premating period (about 89% of the control value at the end of the premating phase). During gestation and lactation of F2 litter, mean body weights of the high dose F1 dams were statistically significantly lower than the corresponding control values. During premating, gestation and lactation periods body weight gain of the high dose females reached or even exceeded body weight gain of the controls.
The statistically significantly lower body weights recorded for the 5000 ppm F1 males and females were considered to be related to the test substance administration. A lower body weight was also recorded for these animals at the pup stage; during the following premating period the F1 parental animals of the high dose group gained substantially as much weight as the controls, but the body weights of the 5000 ppm rats were still reduced. All differences between the controls and 500 or 2500 ppm groups concerning body weights/weight gains, however, were regarded as spontaneous in nature.
 
MALE REPRODUCTIVE FUNCTION
For all F1 males which were placed with females to generate F2 pups, mating was confirmed. The male mating index was 100% in all groups.
 
FEMALE REPRODUCTIVE FUNCTION
The female mating index reached 100% in all groups. The mean duration until sperm was detected (day 0 pc) varied between 2.1 and 3.2 days and was highest for the control group, because one dam of this group had a prolonged cohabitation time. In the scheduled mating interval (F2), 2 control females did not become pregnant. Therefore, the fertility index was lowest in the control group (92%), whereas it was 100% in all substance-treated groups. There were no biologically relevant differences between test groups and the controls concerning the mean duration of gestation and the number of liveborn and stillborn F2 pups. All pregnant females - except one low dose female which had palpable pups in the uterus but did not deliver - gave birth to litters with liveborn pups. Consequently, the gestation and the live birth indices were not influenced by the administration of the test substance. The mean number of delivered pups/dam was not influenced by the test substance administered.

GROSS AND HISTOPATHOLOGICAL FINDINGS
- Thickening of the limiting ridge (margo plicatus) of the forestomach in most male and female rats.
- Minimal hyperkeratosis at the limiting ridge of the forestomach in most male and all female rats.
- Edema in the submucosa of the glandular stomach of 2 male and 10 female rats, minimal in all cases.


Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
240 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: general toxicity
Dose descriptor:
NOAEL
Effect level:
460 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: fertility
Remarks on result:
other: Generation: P and F1 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

**** F1 GENERATION PUPS/LITTERS ****

VIABILITY
No substance-induced effects on pup mortality/viability were recorded during the lactation period. Both the viability index, as an indicator of the viability of the pups during the first 4 days after birth, and the lactation index, as an indicator how the pups were nursed during the rest of their rearing, do not show differences of biological relevance.
 
SEX RATIO
The sex distribution and sex ratios of live F1 pups on the day of birth and on day 21 post parturition (pp) did not show any substantial difference between controls and treated groups; all differences observed are regarded as spontaneous.
 
BODY WEIGHT
Mean body weights of F1 male and female pups were clearly reduced in 5000 ppm test group from day 14 pp onwards and impaired in the intermediate dose (2500 ppm) on day 21 pp when compared to the controls. On day 21 pp the pup weights (both sexes combined) in the high dose group were about 35% and those of the 2500 ppm pups about 11% lower than the corresponding control values. Body weight gains of the 2500 ppm and 5000 ppm pups were also statistically significantly decreased from days 7 (5000 ppm) or 14 pp (2500 ppm) up to weaning (day 21 pp). The reductions in pup body weights/body weight gains in the 5000 and 2500 ppm groups were attributed to the test substance administration. All other differences concerning pup body weights/body weight gains are without any biological relevance and lie within the biological range of variation.
 
CLINICAL OBSERVATIONS
- None of the F1 pups of any one group showed abnormal clinical findings during the lactation period.
- There were no biologically relevant differences between the control and the substance-treated F1 pups in the several morphological development stages monitored up to weaning.
- No remarkable differences between the groups were observed in the different behavioral tests which the pups underwent up to weaning.
- Only spontaneous findings were seen at necropsy (e.g. incisors sloped, hernia diaphragmatica, dilated renal pelvis) in very few of the pups examined. All findings were present in the concurrent control at a comparable frequency and/or did not show a clear relation to dosing.



**** F2 GENERATION PUPS/LITTERS ****

PUP NUMBER AND STATUS
The mean number of delivered F2 pups/dam in the treatment groups was similar to the relevant control value; moreover, the percentages of liveborn and stillborn F2 pups were comparable between the groups; all differences between the groups are in the range of biological variation.
 
VIABILITY/MORTALITY
During the lactation period a statistically significant increase in pups cannibalized by dams was noted in the high and intermediate dose groups. The increased cannibalization rate was predominantly caused by just one intermediate dose dam and two high dose dams. One Female of the high group, however, neglected her pups during the lactation period, thus nearly all pups of this dam died before schedule and/or were cannibalized. Occasionally insufficient nursing behavior and cannibalism occured also in control females, and thus the higher rate of cannibalized pups at these dose levels was not considered treatment-related.
There were no differences in biological relevance between the control and the 500, 2500 and 5000 ppm F2 pups concerning viability and mortality; consequently the viability and lactation indices were not affected by the test substance administration (although some statistically significant differences existed). All relevant values are inside the historical control range and/or do not show a clear relation to dosing; moreover it had to be taken into consideration, that the high dose dams delivered on average distinctly more pups (14.0 pups/dam) than the controls (12.9 pups/dam).
 
SEX RATIO
No remarkable differences between the control and the substance-treated groups were found in respect to the sex ratio of the F2 pups. The observable differences are in the range of biological variation.
 
BODY WEIGHT
Mean body weights/body weight gains of the F2 male and female pups of the 5000 ppm and 2500 ppm groups were clearly influenced by the test substance administration. Mean pup body weights of the 5000 ppm pups were statistically significantly lower than the corresponding control values from days 14 (males and females) until weaning on day 21 pp, when the high dose pups (both sexes combined) weighed about 32% less than the controls. Mean pup body weights of the 2500 ppm pups were statistically significantly (about 12%) lower than the corresponding control values on day 21 pp (both sexes combined). Weight gains of the pups of the 2500 and 5000 ppm test groups were also statistically significantly reduced from the second week of the lactation period onward, the reduction more pronounced in the 5000 ppm than in the 2500 ppm pups. All differences between the control group and the 500 ppm group concerning pup body weight data of the F2 generation were considered spontaneous in nature.
 
CLINICAL OBSERVATIONS
- F2 generation pups did not show any clinical signs up to weaning which could be attributed to the treatment. Hydrocephaly, which occurs also occasionally in control pups was recorded in one 500 ppm pup.
- Development stages: There was a statistically significantly lower incidence of F2 pups/litter with auditory canal opening on time in the intermediate dose group and with eye opening on time in the 5000 ppm group. The relevant values were within the historical control ranges and a clear relation to dosing was not observed. These effects must be considered in conjunction with the retarded weight gain of these pups and were therefore assessed as being possibly substance-related. There were no differences of biological relevance in different stages of development between the low dose and the control pups.
- No substantial differences could be noted between the F2 pups of all test groups and the control pups in the different behavioral tests. The observable differences were without biological relevance.
- The examinations of F2 pups at necropsy did not reveal any differences considered to be of biological relevance between the controls and the substance-treated groups either in the type or in the number of pup necropsy observations. A few pups of the different groups showed some spontaneous findings like hernia diaphragmatica, incisors sloped, dilated renal pelvis, hydroureter, hydrocephaly, focal liver necrosis, cardiomegaly, septal defect and post mortem autolysis.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
53 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: general toxicity

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
53 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: general toxicity

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Mean Body Weight Changes (F0 parental animals), grams

Treatment week

 0 mg/kg bw/d

 53 mg/kg bw/d

 240 mg/kg bw/d

 460 mg/kg bw/d

male

female

male

female

male

female

male

female

0 - 1

52.6

23.8

52.2

24.8

52.3

23.6

46.7**

23.1

1 - 2

53.7

22.1

55.5

22.0

54.4

22.3

51.6

21.8

2 - 3

47.2

16.2

45.8

17.2

46.9

17.5

44.8

14.6

3 – 4

35.4

12.9

34.8

17.1*

35.1

14.9

32.4

17.8*

4 – 5

29.1

15.7

27.6

13.3

28.6

12.6

27.0

13.7

5 – 6

21.0

9.4

21.8

8.8

23.3

12.5

23.2

11.0

6 – 7

23.2

8.5

24.7

11.7

21.2

10.1

19.3*

10.7

7 – 8

20.4

7.7

17.7

10.3

19.8

7.8

18.5

8.6

8 – 9

19.3

9.8

19.1

7.1

18.0

7.2

16.6

8.1

9 – 10

13.1

4.7

13.0

4.5

14.6

7.9*

11.9

6.0

10 – 11

-7.0

 

-2.8

 

-2.8

 

-2.8

 

11 – 12

21.8

 

18.5

 

18.4

 

14.7**

 

12 – 13

14.8

 

13.3

 

13.7

 

11.1

 

13 – 14

8.0

 

10.7

 

9.7

 

6.1

 

14 – 15

9.2

 

7.0

 

7.5

 

8.1

 

15 – 16

10.5

 

10.4

 

8.0

 

9.9

 

16 – 17

5.7

 

7.9

 

7.3

 

4.6

 

17 – 18

2.7

 

4.6

 

1.7

 

1.4

 

18 – 19

1.1

 

0.2

 

1.3

 

1.6

 

19 - 20

4.1

 

6.7

 

5.8

 

5.0

 

*P<0.05

**P<0.01

Reproduction and litter data for F0 parents /F1 pups

 

0 ppm

500 ppm

2500 ppm

5000 ppm

Parents

Females mated

25

25

25

25

Females pregnant

24

23

23

24

Females with delivery

24

23

23

24

Mean duration of gestation (d)

22.0

21.9

21.9

21.9

Litter means

Live births/litter

13.8

13.8

13.7

14.3

Survivors day 4 preculling

13.3

13.5

13.4

13.8

Survivors day 4 postculling

7.5

7.9

7.9

7.9

Survivors day 21

7.5

7.9

7.8

7.8

Weight at day 1 (g) M/F

6.4/6.1

6.6/6.2

6.5/6.2

6.5/6.2

Weight at weaning (g) M/F

52.3/50.01

52.1/49.4

46.6**/44.6**

34.2**/32.7**

Sex ratio of live newborns % M/F

51/49

49/51

55/45

53/47

Selected as parents for the next generation M/F

25/25

25/25

25/25

25/25

**P≤0.01

Reproduction and litter data for F1 parents /F2 pups

 

0 ppm

500 ppm

2500 ppm

5000 ppm

Parents

Females mated

25

25

25

25

Females pregnant

23

23

23

24

Females with delivery

23

23

23

24

Mean duration of gestation (d)

22.0

21.9

21.9

21.9

Litter means

Live births/litter

12.5

11.6

12.0

13.8

Survivors day 4 preculling

11.8

10.6

10.8

12.5

Survivors day 4 postculling

7.7

7.0

7.5

7.8

Survivors day 21

7.7

6.9

7.5

7.4

Weight at day 1 (g) M/F

6.3/5.9

6.5/6.2

6.1/5.8

6.4/6.1

Weight at weaning (g) M/F

50.4/48.4

52.1/49.4

44.6**/42.4**

34.5**/33.2**

Sex ratio of live newborns % M/F

47/53

55/45

53/47

49/51

**P≤0.01

Development landmarks in the F2 (mean % of pups reaching criteria/litter)

Parameter

 

0 ppm

500 ppm

2500 ppm

5000 ppm

Historical control range

Pinna unfolding

93.6 (16.65)

93.5 (20.87)

80.7 (33.37)

86.9 (26.87)

74-100

Auditory canal opening

98.8 (3.87)

95.1 (20.90)

91.4*(18.09)

94.2 (12.26)

81-100

Eye opening

93.2 (18.08)

92.4 (21.89)

92.4 (21.89)

86.5*(21.15)

85-100

*P≤0.05

Figures in parentheses indicate standard deviations.

Applicant's summary and conclusion