Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Oct 2009 - 08 Nov 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study conducted in compliance with GLP regulations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tris-(2-ethylhexyl)amin O 2446
- IUPAC name: 2-Ethyl-N,N-bis(2-ethylhexyl)-hexylamine
- Physical state: liquid / colorless, clear
- Analytical purity: 99.7 corrected area-%, corrected for the water content (BASF Analytical Report, study code 09L00245)
- Purity test date: 27 Oct 2009
- Lot/batch No.: Behälter 407 v. 16.07.2009
- Stability under test conditions: The stability under storage conditions was confirmed by reanalysis.
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar, Crl:WI(Han)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11 - 13 weeks old
- Weight at study initiation: male animals: 281.2 g - 307.2 g; female animals: 180.1 g - 211.2 g
- Housing: individually in Makrolon type M III cages, except during overnight matings, when male and female mating partners were housed together.
- Diet (ad libitum): Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: about 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15 times
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on mating procedure:
- M/F ratio per cage: In general, each of the male and female animals was mated overnight in a 1 : 1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group.
- Length of cohabitation: 2 weeks at maximum
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The following analytical verifications of the stability of the test substance in the diet were carried out prior to the start of the study or during the study:
• 14 days at room temperature
• 42 days storage in refrigerator
• 15 days storage in refrigerator and subsequently 0, 7, 14 and 27 days at room temperature
Homogeneity and concentration control analyses were carried out at the beginning of the premating period and during gestation period.

All measured values for Tris-(2-ethylhexyl)amin O 2446 were in the expected range of the target concentrations (90 - 110 %), demonstrating the correctness of the diet preparations.
Duration of treatment / exposure:
Females: 51 or 53 days
Males: 31 days
Frequency of treatment:
continuously in the diet until 16 hours before sacrifice
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1500, 4000, and 12000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
125, 327, and 900 mg/kg bw/d
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
about 105, 277, and 815 mg/kg bw/d in males; about 103, 271, 770 mg/kg bw/d in non-pregnant females; about 121, 306 and 918 mg/kg bw/d in pregnant females; about 173, 455, 1098 mg/kg bw/d in lactating females
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed clinical observation (DCO) was performed in all animals once before the first administration and thereafter at weekly intervals.
- For observation, the animals were removed from their cages by the investigator and placed in a standard arena for at least 20 seconds/animal (50 x 37.5 cm wide, with side borders which are 25 cm high).
For details see 7.5.1 Repeated dose toxicity: oral

BODY WEIGHT: Yes
- Time schedule for examinations: once a week
- The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND 1) and on PND 4.
Body weight was not determined in females showing no positive evidence of sperm during mating and gestation periods or in females without litter during lactation period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male F0 animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined for gestation days (GD) 0 - 7, 7 - 14 and 14 - 20.
• Food consumption of F0 females, which gave birth to a litter was determined for PND 1- 4.
Food consumption was not determined in females without positive evidence of sperm during mating and gestation periods and in females without litter during lactation period.

The intake of test substance was calculated from the amount of food consumed and expressed in mg/kg body weight per day.
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Ovaries
10. Pituitary gland
11. Prostate
12. Seminal vesicles with coagulation glands
13. Spleen
14. Testes
15. Thymus
16. Thyroid glands
17. Uterus with cervix

HISTOPATHOLOGY: Yes
The following organs or tissues were fixed in 4% formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Coagulation glands
8. Colon
9. Duodenum
10. Epididymides (modified Davidson’s solution)
11. Esophagus
12. Eyes with optic nerve
13. Female mammary gland
14. Femur with knee joint
15. Heart
16. Ileum
17. Jejunum (with Peyer’s patches)
18. Kidneys
19. Larynx
20. Liver
21. Lungs
22. Lymph nodes (mesenteric and axillary lymph nodes)
23. Nose (nasal cavity)
24. Ovaries (modified Davidson’s solution)
25. Oviducts
26. Pancreas
27. Pharynx
28. Pituitary gland
29. Prostate
30. Rectum
31. Salivary glands (mandibular and sublingual glands)
32. Seminal vesicle with coagulation glands
33. Sciatic nerve
34. Skeletal muscle
35. Skin
36. Spinal cord (cervical, thoracic and lumbar cords)
37. Spleen
38. Sternum with marrow
39. Stomach (forestomach and glandular stomach)
40. Testes (modified Davidson’s solution)
41. Thymus
42. Thyroid glands/parathyroid glands
43. Trachea
44. Urinary bladder
45. Uterus
46. Vagina
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings. All hematoxylin-eosin embedded tissues from the control and high-dose group animals were assessed (in most cases from 5 animals/sex/group).
Statistics:
- Body weight and body weight change of pups (litter means), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter: DUNNETT-test (two-sided)
- Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at
necropsy: FISHER'S EXACT test
- Proportions of affected pups per litter with necropsy observations: WILCOXON-test (one-sided)
Reproductive indices:
- Male mating index (%) = (number of males with confirmed mating / number of males placed with females) X 100
- Male fertility index (%) = (number of males proving their fertility / number of males placed with females) X 100
- Female mating index (%) = (number of females mated / number of females placed with males) X 100
- Female fertility index (%) = (number of females pregnant / number of females mated) X 100
- Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant) X 100
- Live birth index (%) = (number of liveborn pups at birth / total number of pups born) X 100
- Postimplantation loss (%) = number of implantations – number of pups delivered / number of implantations) X 100
Offspring viability indices:
- Viability index (%) = (number of live pups on day 4 after birth / number of live pups on the day of birth) X 100
- Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) X 100

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no test substance-related or spontaneous mortalities in any of the groups.
No clinical signs or changes of general behaviour, which may be attributed to the test substance, were detected in any male or female F0 generation parental animals during the whole study including gestation and lactation periods.
One sperm positive control female, one sperm positive low-dose female, three sperm positive mid-dose, and two high-dose females did not deliver F1 pups.

BODY WEIGHT (PARENTAL ANIMALS)
Body weights of the high-dose females (12000 ppm) were statistically significantly lower during gestation (9 % below control on GD 20) and during lactation (PND 1 weight 8 % and PND 4 weight 7 % below control). Body weight gain of the high-dose females was statistically significantly lower than control during gestation (GD 7 - 20 up to about 37 %, GD 0 - 20 about 25 %). Body weights and body weight gain of all treated males were unchanged throughout the study as were body weight/ body weight gain of the low- and mid-dose females. This statement includes the statistically significantly increased body weights of the low- and mid-dose females during premating week 2.

FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption of the high- and mid-dose F0 males (12000 or 4000 ppm) was slightly but statistically significantly below control during premating weeks 0 - 1 (about 8 % or 6 %). Low-dose males (1500 ppm) did not show any test substance-related changes of food consumption.
Food consumption of the high-dose F0 females was statistically significantly below control between GD 14 - 20 (about 13 %) and between PND 1 - 4 (about 27 %). High-dose F0 females during premating and mid and low-dose females during all study phases did not show any test substance-related changes of food consumption.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
MALES:
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100 % in all groups including the controls.
Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
One control male, one low-dose male, two mid-dose males and one high dose male did not generate F1 pups. No histomorphological correlate was found to explain these apparent infertilities. Thus, the male fertility index ranged between 80 % and 90 % without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

FEMALES:
The female mating index for F1 litter was 100% in all test groups.
The mean duration until sperm was detected (GD 0) varied between 2.4 and 6.0 days without any relation to dosing. The unexpected high control value of 6.0 days, which is outside historical control range (2.1 - 5.3 days), was caused by three females which had no sperm in vaginal smear until day 13.
All sperm positive rats delivered pups or had implants in utero with the following exceptions:
• one control female did not become pregnant.
• one low-dose female did not become pregnant.
• two mid-dose females did not become pregnant.
• one high-dose female did not become pregnant.
The fertility index varied between 80 % in test group 2 (4000 ppm) and 90 % in control and test groups 1 and 3 (0, 1500 and 12000 ppm). These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
The non-pregnant female rats did not show a histomorphological correlate to explain the apparent infertilities.
The mean duration of gestation was similar in all test groups (i.e. between 21.7 and 22.0 days).
The gestation index varied between 88 % in test group 2 (4000 ppm), 89 % in test group 3 (12000 ppm) and 100 % in the control and test group 1 (1500 ppm).
The mean number of implantations was not statistically significantly different from concurrent control in all dose groups. However, the average numbers of implantations were below the historical range for this parameter in all dose groups. Within dosed groups, individual implantation numbers showed high variability at mid- and high-dose levels, but not at low-dose level. Correspondingly, the average litter size showed no statistically significant differences between dosed groups and control, although all values in treated groups were below concurrent control and for the high-dose group the average litter size was below historical control. Postimplantation loss was higher than control in the mid and high-dose groups, although the difference was not statistically significant.
The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 100 % (control and all test groups). Moreover, no stillborn pups were observed in any of the groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Statistically significant weight differences recorded for the ovary, spleen, adrenal gland, liver and kidney were considered to be test item-related.
Most prominent organ weight changes measured in this study were in the ovaries. Corroborating the macroscopic and histopathological observations, mean absolute and relative ovary weights were statistically significantly higher in all treated female groups. The effect was dose-related. Mean absolute and relative spleen weights were statistically significantly higher in all treated female groups, the difference to controls being strongest in the group administered 4000 ppm. No effect was noted on spleen weights of the males. Mean absolute adrenal gland weights were higher in both sexes administered 4000 ppm, and mean relative adrenal gland weights were higher in both sexes administered 4000 or 12000 ppm. The difference to controls was minor in severity and lacked a dose relationship. Mean absolute and relative liver weights were statistically significantly higher in the female group administered 4000 ppm. In the females treated at 12000 ppm, only the relative liver weight was statistically significantly increased. The effect was minor in severity and was not seen in the males. Mean absolute kidney weights were statistically significantly higher in females administered 1500 or 4000 ppm, and mean relative kidney weights were statistically significantly higher in severity and not dose-related. There was no effect on kidney weights of the males. Some other statistically significant organ weight differences to controls were recorded in this study. Mean absolute and relative epididymis weights were minimally higher in males treated at 4000 ppm, but this variation was considered incidental in the view of its low magnitude and lack of dose relationship. Compared to controls, mean absolute heart weight was minimally lower and mean relative brain weight was minimally higher in the female group administered 12000 ppm. This difference was interpreted to be a consequence of the lower mean terminal body weight recorded in this group and thus not to be directly treatment-related. All other organ weight variations noted in this study were not statistically significant and considered to be fortuitous.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At terminal necropsy, one female of the control group, one female treated at 1500 ppm, two females treated at 4000 ppm and one female treated at 12000 ppm were found not to be pregnant.
Macroscopic organ changes associated with histopathological lesions and therefore considered to be test item-related were seen in the females. Corresponding to organ weight changes, enlarged ovaries were seen in a dose-related manner, in females of all three treated groups. Spleen and/or mesenteric lymph node were enlarged in occasional females treated at 4000 or 12000 ppm. In single females of the high dose group, only, axillary lymph nodes, mediastinal lymph nodes or liver lymph node were enlarged. Also, prominent liver foci in one female administered 12000 ppm were interpreted to be test item-related in view of the corroborative histopathological changes seen.
Other macroscopic organ findings were very few and considered to be incidental and not treatment-related.

HISTOPATHOLOGY (PARENTAL ANIMALS)
For histopathological findings in other than reproductive organs see Chapter 7.5.1.
REPRODUCTIVE ORGANS
Ovary
In the corpora lutea of the ovary, a foamy change with mixed cell inflammation was observed in all test item-treated females, the mean severity of the change being clearly dose-related.

No other histopathological lesion considered to be test item-related was noted in the male or female reproductive organs.
At terminal necropsy, one control female and four test item-treated females were found not to be pregnant. However, no dose relationship was observed for this finding, and no relevant histopathological findings were seen in mates of the females concerned. Therefore, occasional failure of pregnancy could not be attributed to the treatment in the present study.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
900 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects on fertility of the F0 parental animals of both sexes.

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
The mean number of delivered F1 pups per dam (average litter size) showed no statistically significant differences between dosed groups and control, although all values in treated groups were below concurrent control and for the high-dose group the average litter size was below historical control. The rates of liveborn and stillborn F1 pups were evenly distributed about the groups, the respective values reflect the normal range of biological variation inherent in the strain used in this study.
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 97 % (test group 3), 99 % (test group 2) and 100 % (control and test group 1).
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

CLINICAL SIGNS (OFFSPRING)
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

BODY WEIGHT (OFFSPRING)
Mean body weights of the high-dose pups (12000 ppm) were below control on PND 4, the difference was, however, not statistically significant. High-dose female pup weight on PND 4 was outside the historical control range. The average difference to the concurrent control was about 13 % (both sexes combined). The high-dose pups gained about 30 % less weight than the controls. At necropsy 3 high-dose pups had an empty stomach indicating some disturbance of maternal care in this group.
Pup body weights/body weight gain of the low- and mid-dose pups were not influenced by the treatment.
The number of "runts" was highest in the high-dose group, mainly caused by one dam.

GROSS PATHOLOGY (OFFSPRING)
Three pups of one high-dose dam showed an empty stomach at gross necropsy on PND 4. No other findings were seen in any pup of any group.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
327 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Slightly reduced growth and development of offspring, secondary to maternal toxicity.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

DISCUSSION

Tris-(2-ethylhexyl)amin O 2446 was administered as a constant homogeneous addition to the food in different concentrations (1500, 4000 and 12000 ppm) to groups of 10 male and 10 female Wistar rats (F0 animals). These concentrations in feed correspond to about 105, 277 and 815 mg/kg bw/d in males; 103, 271, 770 mg/kg bw/d in non-pregnant females; 121, 306 and 918 mg/kg bw/d in pregnant females; 173, 455, 1098 mg/kg bw/d in lactating females.

 

Clinically, toxicity was noted in the F0 males and F0 females at 4000 and 12000 ppm. Salient clinical findings were decreases in food consumption and body weights/ body weight gain which were most severe in females during gestation and lactation and led to a minimally lower mean terminal body weight in females administered 12000 ppm.

Detailed clinical examinations in an open field, detailed observations in a functional observational battery (FOB) and measurements of motor activity did not reveal any indications of test substance-induced effects in low-, mid- and high-dose rats (1500 ppm [125 mg/kg bw/d], 4000 ppm [327 mg/kg bw/d] and 12000 ppm [900 mg/kg bw/d]).

Clinical pathology revealed that in all dosed female rats the absolute neutrophil and monocyte counts and correspondingly the total white blood cell counts were elevated. This was most probably related to inflammatory changes in various organs.

The increased serum ALT activities in female and male rats of the high dose group (12000 ppm) indicated a slight degradation of the liver cell membranes. The lower albumin levels in females of dose group 2 and 3 (4000 and 12000 ppm) could be interpreted as an altered liver cell metabolism in these animals.

Pathology and histopathology revealed a multitude of test substance-related findings. Treatment led to minimally lower mean terminal body weight in females administered 12000 ppm and to prominent, dose-related ovary weight increases in all treated female groups. Other minor organ weight increases considered to be test substance-related were observed for the spleen, adrenal gland, liver and kidney, predominantly in the females, but were not clearly dose-related.

Test substance-related macroscopic organ changes were seen in the females, only, and mainly comprised enlarged ovaries in all dose groups and enlarged spleen and/or mesenteric lymph node in occasional females treated at 4000 or 12000 ppm.

Predominant histopathological findings were dose-related histiocytic/mixed cell infiltrates in multiple organs, with an overall tendency of being more severe in the females.

In the small intestine, minimal villous infiltrates of histiocytes and/or mixed cells were seen in some males and females treated at 12000 ppm and in single females of the lower dose groups. In individual females, these small intestinal changes were accompanied by minimal focal abscess(es)/necrotic foci and/or mural histiocytic/mixed cell infiltrates. Histiocytic/ mixed cell infiltrates were also seen in the Peyer's patch in a dose-related manner in some females treated at 4000 or 12000 ppm and in one male treated at 12000 ppm. Moreover, dose-related minimal to massive degrees of histiocytic/mixed cell infiltrates were noted in the mesenteric lymph node (draining lymph node of the small intestine) in the majority of males and females of all dose groups, in some animals together with lymphoid hyperplasia, abscesses/necrotic foci and/or adjacent mixed cell infiltrates. The axillary, mediastinal and/or liver lymph node of single females administered 12000 ppm showed similar findings.

In the stomach, minimal or slight degrees of a focally extensive submucosal edema/ inflammation were observed in a proportion of males and females treated at 4000 or 12000 ppm. The urinary bladder of a single female treated at 12000 ppm showed minimal mural histiocytic/mixed cell infiltrates, which were also considered test substance-related.

In the corpora lutea of the ovary, a prominent foamy change with mixed cell inflammation was observed in all test substance-treated females, the mean severity of the change being clearly dose-related.

The liver of one female treated at 12000 ppm showed a moderate degree of histiocytic/mixed cell infiltrates with necrosis, which was considered test substance-related. No histopathological change was found to corroborate the liver weight differences recorded at necropsy.

In the adrenal gland, mixed cell infiltration and single cell death in the zona fasciculata were seen in one female each of the 4000 ppm and 12000 ppm dose group and might be related to test substance administration. Moreover, diffuse cortical hypertrophy was noted in some females administered 4000 or 12000 ppm. This minor change might account for adrenal gland weight changes noted at necropsy and was considered to possibly represent a metabolic adaptation of the adrenal cortex to treatment.

It can be speculated that the infiltrates in the small intestine, draining lymph nodes and other organs might represent histiocytes containing test substance-lipid complexes which are poorly degraded by lysosomal enzymes and lead to a secondary local inflammatory reaction. Ovarian changes might also represent storage of test substance-lipid complexes with inflammation and have been described in the literature to occur after administration of amphophilic cationic chemical entities.

Minimal mixed cell infiltration in the spleen of two females treated at 12000 ppm, as well as increased granulopoiesis in the bone marrow of female dose groups treated at 4000 or 12000 ppm were probably a consequence of the inflammatory small intestinal and lymph node changes.

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats Tris-(2- ethylhexyl)amin O 2446 had no adverse effects on fertility of the F0 parental animals of both sexes at 1500, 4000 and 12000 ppm. Most of the F0 parental animals proved to be fertile. Failure of pregnancy in one control female and in four test substance-treated females could not be attributed to the treatment by gross and histopathological examinations of the respective animals of both genders.

The test substance caused no impairments of most reproductive performance parameters. Mating behaviour, conception and parturition were not influenced. However, mean number of implantations and average litter size were below concurrent control in all treated groups. Postimplantation loss was higher than control in the mid- and high-dose groups only. The average number of implantations was below the historical range in all dose groups and postimplantation loss was above historical control in mid- and high dose groups.

Within dosed groups, individual postimplantation loss data showed high variability at mid and high-dose levels, but not at low-dose level. Particularly, one female each in the mid dose and high-dose groups had only 1 or 2 resorbed implants in the uterus. These individuals had an above-average influence on mean postimplantation loss in these groups. If they were taken out from calculation of mean, the postimplantation loss in the mid-dose group is 3.5% (instead of 15.5%) and in the high-dose group it is 7.7% (instead of 18.0%). Thus the increased average postimplantation loss in the mid- or high-dose group may have been caused by two independent incidental events in two individual females.

Also, individual implantation numbers showed high variability within dosed groups. Other than for postimplantation loss there appears to be a dose-related trend in the incidence of females with below-average implantations: 11% control, 33% low-dose, 38% mid-dose and 56% high-dose pregnant animals. The higher numbers in treated groups may be correlated to the described inflammatory ovarian changes subsequent to storage of test substance lipid complexes in the ovaries. In particular corpora lutea were affected by a prominent foamy change with mixed cell inflammation, thus affecting a gland which is crucial for the preparation of uterine mucosa for implantation. This suggests a relationship of the lower number of implants to the treatment.

The only sign of developmental toxicity was a rather slight reduction of offspring body weights/body weight gain exclusively at the high-dose level of 12 000 ppm. This probably temporary developmental delay is considered to be secondary to the impaired well-being of the mothers in this dose group, as was hinted to by empty stomachs in some individual high dose pups. There were no other findings in the offspring which might indicate a selective influence of the test compound on postnatal development.

 

 

CONCLUSION

Thus, under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for fertility is 12000 ppm (900 mg/kg bw/d) for the F0 parental rats.

No NOAEL for general, systemic toxicity of the test substance was determined for the F0 parental animals based on clinical-pathological and histopathological findings in all treated groups, predominantly dose-related histiocytic/mixed cell infiltrates in multiple organs (ovaries, intestines and their draining lymph nodes) which lead to secondary local inflammatory reactions. These inflammations, above all in the ovaries, presumably also caused a slight adverse effect on reproductive performance as indicated by slightly lower numbers of implants in all treated groups. Females were more sensitive than males.

The NOAEL for developmental toxicity in the F1 progeny of the test substance treated groups was found to be 4000 ppm (327 mg/kg bw/d) toxicity based on slightly reduced growth and development of offspring, secondary to maternal toxicity.

 

Applicant's summary and conclusion