Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

In a screening for reproductive and developmental toxicity according to OECD TG 422 male and female WIstar rats were fed with the the test substance.

The NOAEL for fertility parameters is 12000 ppm (900 mg/kg bw/d) for the F0 parental rats (fertility, mating behaviour, conception and parturition were not influenced).

However, dose-related inflammations were observed in maternal animals, in the ovaries, lymph nodes and intestines. 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 group. Females were more sensitive than males.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
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:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
22 Mar 1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Analytical purity: 99.7%
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Sex:
male/female
Details on test animals or test system 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
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight, maximum of two weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: pregnant animals and their litters housed together until PND 4 in Makrolon type M III cages (Becker & Co., Castrop-Rauxel, Germany
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 the test substance 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
Dose / conc.:
1 500 ppm
Remarks:
equivalent to 125 mg/kg bw/d nominal in diet
equivalent to approx. 105/103/121/173 mg/kg bw/d in males/non-pregnant females/pregnant females/lactating females
Dose / conc.:
4 000 ppm
Remarks:
equivalent to 327 mg/kg bw/d nominal in diet
equivalent to approx. 277/271/306/455 mg/kg bw/d in males/non-pregnant females/pregnant females/lactating females
Dose / conc.:
12 000 ppm
Remarks:
equivalent to 900 mg/kg bw/d nominal in diet
equivalent to approx. 815/770/918/1098 mg/kg bw/d in males/non-pregnant females/pregnant females/lactating females
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Fasting period before blood sampling for clinical biochemistry: 16 hours
Positive control:
not applicable
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:
- 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):
Parameters examined in P male parental generations:
- testis weight, epididymis weight, tubular stages of the spermatogenic cycle (histopathological observations)
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).
Postmortem examinations (offspring):
SACRIFICE
- the F1 offspring animlas were sacrificed at 4 days of age (PND 4).
- pups were examined externally and eviscerated; their organs were assessed macroscopically
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
Clinical signs:
no effects observed
Description (incidence and severity):
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.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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 and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
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.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
For histopathological findings in other than reproductive organs see chapter repreated dose toxicity, same study.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
For histopathological findings in other than reproductive organs see chapter repreated dose toxicity, same study.
Urinalysis findings:
no effects observed
Description (incidence and severity):
For histopathological findings in other than reproductive organs see chapter repreated dose toxicity, same study.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
For histopathological findings in other than reproductive organs see chapter repreated dose toxicity, same study.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Small intestine:
In the small intestine, minimal villous infiltrates of histiocytes and/or mixed cells were seen in the jejunum, duodenum and/or ileum in a small proportion of animals treated at 12000 ppm and in single females treated at 1500 or 4000 ppm (jejunum only). In single females, these small intestinal changes were accompanied by minimal focal abscess(es)/necrotic foci and/or mural histiocytic/mixed cell infiltrates.

Peyer's patch:
In the small intestinal Peyer's patch, histiocytic/mixed cell infiltrates were seen in a dose related manner in some females treated at 4000 or 12000 ppm and in one male treated at 12000 ppm. The occurrence of minimal infiltrates in isolated females of the control group and low dose group was considered to be fortuitous and unrelated to the treatment.

Mesenteric lymph node:
Minimal to massive degrees of histiocytic/mixed cell infiltrates were also seen in the mesenteric lymph node (draining lymph node of the small intestine), in a dose-related manner in males and females of all dose groups, with a tendency of showing higher degrees of severity in the females. These changes were associated with minor lymphoid hyperplasia of the lymph node in a proportion of males and females of all dose groups, and with abscesses/necrotic foci in a proportion of females treated at 12000 ppm and one single female treated at 4000 ppm. Moreover, in a proportion of treated females of all dose groups, mixed cell infiltrates were seen in the tissue adjacent to the lymph node.

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 and were seen mostly at the glandular portion of the stomach.

Liver:
The liver of one female treated at 12000 ppm showed a moderate degree of histiocytic/mixed cell infiltrates with necrosis, confirming the macroscopic observation of liver foci in this animal. This change was considered test item-related. No histopathological change was found to corroborate the liver weight differences recorded at necropsy.

Adrenal gland:
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 item 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.

Spleen:
In the spleen, a minimal mixed cell infiltration was seen in two females treated at 12000 ppm. This was probably a consequence of the inflammatory small intestinal and lymph node changes and therefore considered test item-related.

Bone marrow:
Likewise, in the femoral bone marrow, a minimally or slightly increased myeloid:erythroid ratio was noted in a proportion of females treated at 4000 or 12000 ppm and was considered to represent increased granulopoiesis as a consequence of the inflammatory state in these animals.

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.

Other organs:
In single females administered 12000 ppm, the axillary, mediastinal and/or liver lymph node showed histiocytic/mixed cell infiltrates with or without presence of abscess(es)/necrotic foci, and corresponding to changes noted in the mesenteric lymph node. These lesions were therefore considered test item-related. The urinary bladder of a single female treated at 12000 ppm showed minimal mural histiocytic/mixed cell infiltrates, which were also considered test item-related. In the kidney, no histopathological changes were found to account for the higher kidney weights noted in the test item-treated groups.
A number of other histopathological findings were noted in treated and/or control rats, but were considered to be most likely incidental and/or to be within the range of expected changes for Crl:WI(Han) rats of this age kept under experimental conditions.

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.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
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.
Dose descriptor:
NOAEL
Remarks:
No adverse effects on fertility of the F0 parental animals of both sexes.
Effect level:
12 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: equivalent to 900 mg/kg bw/d
Key result
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
reproductive performance
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day (nominal)
System:
other: histiocytosis in various organs
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
Mortality / viability:
no mortality observed
Description (incidence and severity):
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).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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.
Sexual maturation:
no effects observed
Description (incidence and severity):
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.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
327 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: 327 mg/kg bw = 4000 ppm = mid dose group
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
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 multiple 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.
Executive summary:

SUMMARY

METHODS

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; and 173, 455, 1098 mg/kg bw/d in lactating females, respectively. The control group, consisting of 10 male and 10 female Wistar rats, was kept on basal diet only. The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and approximately 1 week thereafter in females.

OBSERVATIONS

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females.

A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals.

Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4.

Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, after the day of parturition (postnatal day [PND] 1) and on PND 4.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed one PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings.

Clinicochemical and hematological examinations as well as urinalyses were performed in 5 parental animals of either sex towards the end of the administration period.

At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group.

All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

RESULTS

Analytics

The various analyses confirmed:

• the stability of the test substance preparations in diet for a period of 7 days at room temperature and for a period of 42 days stored in a refrigerator

• the homogeneous distribution of the test substance in the diet

• the correctness of the prepared concentrations

Effects

The following salient test substance-related adverse effects/findings were noted:

Test group 3 (12000 ppm [900 mg/kg bw/d])

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

• Reduced food consumption in males (week 0 – 1, 8% below controls) and in females (GD 14-20, 13% below controls and PND 1 - 4, 27% below controls)

• Lower body weights in females (GD 20, 9% below controls; PND 1, 8% and PND 4, 7% below controls, terminal weight 6% below controls)

• Lower body weight gain in females during gestation (GD 7 – 20, 37% and GD 0 – 20, 25% below controls)

• Increased ALT activities in rats of both sexes

• Increased total white blood cell counts, absolute neutrophil and absolute monocyte counts in females

• Decreased albumin levels in females

• Increased spleen, adrenal, liver and kidney weights in females

• Enlarged spleen and/or mesenteric lymph node in females

• Histiocytic/mixed cell infiltrates in multiple organs (mainly ovaries, intestines and their draining lymph nodes, liver, spleen, adrenals, bone marrow) indicative of inflammatory changes

• Minimal or slight degrees of a focally extensive submucosal edema/ inflammation in stomach

• Markedly increased ovary weights (relative weights 212% of control), macroscopical organ enlargement, histopathologically prominent foamy change with mixed cell inflammation in corpora lutea (severity marked 7/10, moderate 3/10 females)

• Lower average number of implants (8.4 vs. 11.2 in controls)

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

• Slight reduction of body weights/body weight gain, empty stomach in 3 pups

Test group 2 (4000 ppm [327 mg/kg bw/d])

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

• Reduced food consumption in males (week 0 – 1, 6% below controls)

• Increased total white blood cell counts, absolute neutrophil and absolute monocyte counts in females

• Decreased albumin levels in females

• Increased spleen, adrenal, liver and kidney weights in females

• Enlarged spleen and/or mesenteric lymph node in females

• Histiocytic/mixed cell infiltrates in multiple organs (mainly ovaries, intestines and their draining lymph nodes, adrenals, bone marrow) indicative of inflammatory changes

• Minimal or slight degrees of a focally extensive submucosal edema/ inflammation in stomach

• Increased ovary weights (relative weights 183% of control), macroscopical organ enlargement, histopathologically prominent foamy change with mixed cell inflammation in corpora lutea (severity marked 3/10, moderate 7/10 females)

• Lower average number of implants (8.8 vs. 11.2 in controls)

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

• No test substance-related adverse findings

Test group 1 (1500 ppm [125 mg/kg bw/d])

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL

PATHOLOGY/ PATHOLOGY

• Increased total white blood cell counts, absolute neutrophil and absolute monocyte counts in females

• Increased spleen weights in females

• Histiocytic/mixed cell infiltrates in multiple organs (mainly ovaries, intestines and their draining lymph nodes) indicative of inflammatory changes

• Increased ovary weights (relative weights 156% of control), macroscopical organ enlargement, histopathologically prominent foamy change with mixed cell inflammation in corpora lutea (severity moderate 6/10, slight 4/10 females)

• Lower average number of implants (9.9 vs. 11.2 in controls)

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

• No test substance-related adverse findings

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 multiple 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.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
125 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a GLP guideline study according to OECD 422, the test substance was administered as a constant homogeneous addition to the food at concentrations of 1500, 4000 and 12000 ppm to groups of 10 male and 10 female Wistar rats. 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 and 173, 455, 1098 mg/kg bw/d in lactating females. The control group, consisting of 10 male and 10 female Wistar rats, was kept on basal diet only. The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and approximately 1 week thereafter in females. Maternal 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. 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. Predominant histopathological findings were dose-related histiocytic/mixed cell infiltrates in multiple organs (ovaries, intestines and their draining lymph nodes), with an overall tendency of being more severe in the females. 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 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 highdose 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. Thus the increased average postimplantation loss in the mid- or highdose 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.

Thus, the NOAEL for fertility parameters is 12000 ppm (900 mg/kg bw/d) for the F0 parental rats. No NOAEL for general, systemic toxicity 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. Similar effects were also observed in a subchronic toxicity study (see also chapter repeated dose toxicity).

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. 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).

Effects on developmental toxicity

Description of key information

The test substance was tested for its prenatal developmental toxicity in Wistar rats (OECD 414). The test substance was administered as an oily solution to groups of 25 time-mated female Wistar rats by gavage at doses of 50, 150 and 500 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The no observed adverse effect level (NOAEL) for maternal toxicity is 150 mg/kg bw/d. There were no toxicologically relevant adverse fetal findings evident. Thus, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 500 mg/kg bw/d.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-03-04 until 2015-04-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 Jan 2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Germany
- Purity: 99.7 %
- Age at study initiation: about 10-12 weeks
- Weight at study initiation: body weight of the pregnant animals on day 0 varied between 151.5 – 195.7 g
- Housing: individually (Makrolon type M III cages)
- Diet: ad libitum (Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light):12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the test substance preparations the specific amount of test substance was weighed, topped up with corn oil in a graduated flask and intensely mixed by shaking until it is dissolved.

DOSING SOLUTIONS PREPARATION
- Rate of preparation of dosing solution: at the beginning of the administration period and thereafter at intervals
- Storage temperature of dosing solution: room temperature

VEHICLE
- Justification for use and choice of vehicle: not specified
- Amount of vehicle: 4 mL/kg bw/d (substance in vehicle)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
Analytical verifications of the stability of the test substance in corn oil over a period of 7 days at room temperature were conducted prior to the start of the study.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug / sperm referred to as day 0 of pregnancy
Duration of treatment / exposure:
GD 6-19
Frequency of treatment:
daily
Duration of test:
On GD 20, the females were sacrificed in a randomized order and examined macroscopically. The fetuses were removed from the uterus and investigated.
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day on working days or once a day on Saturdays, Sundays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption: The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined/Tissue fixed:
1. All gross lesions
2. Adrenal glands
3. Kidneys
4. Liver
5. Mesenteric lymph nodes
6. Ovaries
7. Spleen
8. Stomach
9. Small intestine
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: No
Statistics:
DUNNETT-test (twosided); FISHER'S EXACT test (onesided); WILCOXON-test (onesided); KRUSKAL-WALLIS test
Indices:
The conception rate (in %) was calculated according to the following formula:
number of pregnant animals/number of fertilized animals x100

The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
number of corpora lutea – number of implantations/number of corpora lutea x 100

The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
number of implantations – number of live fetuses/number of implantations x100
Historical control data:
Was used to assess whether observed differences were within historical control range.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Some females (7 out of 25) of the high-dose group (500 mg/kg bw/d) and some females (5 out of 25) of the mid-dose group (150 mg/kg bw/d) showed transient salivation towards the end of the administration period. Salivation persisted in the respective animals for a few minutes after daily gavage dosing (i.e. up to 10 minutes) and was initially observed on GD 15.
No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 50, 150 or 500 mg/kg bw/d during the entire study period.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 50, 150 or 500 mg/kg bw/d).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights and the average body weight gain of the low-, mid- and high-dose dams (50, 150 and 500 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period. This includes the statistically significant increased body weight gain value in test group 3 on GD 8-10.
The corrected body weight gain of test groups 1-3 (50, 150 and 500 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights remained also unaffected by the treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of the dams in test groups 1, 2 and 3 (50, 150 and 500 mg/kg bw/d) was generally comparable to the concurrent control group throughout the study. This includes the slightly, but statistically significantly increased food consumption values in test group 2 (150 mg/kg bw/d) on GD 15-17 and in test group 3 (500 mg/kg bw/d) on GD 3-6 and GD 15-17.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In dams of test group 3 (500 mg/kg bw/d) red blood cell (RBC) counts, hemoglobin and hematocrit values were decreased. Hemoglobin values were already lower compared to controls in rats of test group 2 (150 mg/kg bw/d). However, in this test group this was the only altered red blood cell parameter and the mean was within the historical control range (hemoglobin 6.3-7.7 mmol/L). Therefore, in this test group the reduced hemoglobin value was regarded as incidental and not treatment-related. In dams of test groups 2 and 3 (150 and 500 mg/kg bw/d), total white blood cell counts, absolute lymphocyte, monocyte and large unstained cell (LUC) counts were increased. However all values apart from the absolute monocyte counts in test group 3 (500 mg/kg bw/d) were within historical control ranges (WBC 3.58-6.00 Giga/L; absolute lymphocyte counts 1.89- 3.85 Giga/L; absolute monocyte counts 0.10-0.15 Giga/L; absolute LUC counts 0.01-0.03 Giga/L). Therefore, the changes of the total white and the differential blood cell counts were regarded as incidental and not treatment-related. The mean of the absolute monocyte counts in test group 3 which was marginally above the historical control range was also regarded at least as not adverse.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In dams of test groups 1, 2 and 3 (50, 150 and 500 mg/kg bw/d) alanine aminotransferase (ALT) activities were higher compared to controls. In test groups 1 and 2 the increase was marginal (less than 2fold); thus the alteration was regarded as treatment-related, but not adverse. In dams of test group 3 (500 mg/kg bw/d) cholesterol and triglyceride values were increased. Cholesterol levels were already higher in test group 2 (150 mg/kg bw/d), but this was the only relevantly changed clinical chemistry parameter in this test group and therefore it was regarded in this test group as treatment-related but not adverse (ECETOC Technical report No. 85, 2002).
In dams of test groups 1, 2 and 3 (50, 150 and 500 mg/kg bw/d), total bilirubin values were decreased. A hypoplastic anemia as cause for this decrease is not probable because of the short application time of the compound for 14 days. More probably an increased conjugation rate in the liver cells coupled with an accelerated excretion via bile was the cause for the lower bilirubin values. This effect was regarded as treatment-related but not adverse.
In dams of test groups 1, 2 and 3 (50, 150 and 500 mg/kg bw/d), calcium levels were higher compared to controls, but the values were within the historical control range (calcium 2.36- 2.68 mmol/L). In dams of test group 1 (50 mg/kg bw/d) total protein and albumin levels were higher compared to controls, but the change was not dose-dependent. Therefore, the mentioned alterations were regarded as incidental and not treatment-related.
Details are given table 1.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The mean gravid uterus weights of the animals of test group 1-3 (50, 150 and 500 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.
The mean placental weights of the low-, mid- and high-dose groups (50, 150 and 500 mg/kg bw/d) were comparable to the corresponding control group.
Details are given in table 2.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred only individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: non-neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 50, 150 and 500 mg/kg bw/d) in the mean number of implantation sites or in the the number of resorptions.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 50, 150 and 500 mg/kg bw/d) in the mean number of implantation sites and in the values calculated for the pre- and the postimplantation losses.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 50, 150 and 500 mg/kg bw/d) in viable fetuses.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The conception rate was 92% in the low-dose group (50 mg/kg bw/d) and 100% in the control, mid- and high-dose groups (0, 150 and 500 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of the study. There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 50, 150 and 500 mg/kg bw/d) in conception rate.
Other effects:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 50, 150 and 500 mg/kg bw/d) in the mean number of corpora lutea.
All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical biochemistry
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
other: No adverse effects on fetuses.
Abnormalities:
effects observed, treatment-related
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean fetal weights of test groups 1-3 (50, 150 and 500 mg/kg bw/d) were not influenced by the test substance. This includes the statistically significantly higher mean fetal body weight in test group 3, which was well within the historical control range (both sexes combined: mean 3.5 g [2.5 – 5.1 g]). Therefore it was regarded as incidental and not treatment-related.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (50, 150 and 500 mg/kg bw/d) was comparable to the control fetuses.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
An external malformation was recorded, each, for one control fetus and one fetus of test group 3 (500 mg/kg bw/d), as shown in table 3. For the affected fetuses, these external findings were associated either with skeletal or visceral malformations. The total incidence of external malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.
No external variations were recorded.
One unclassified external observation, i.e. blood coagulum around placenta, was recorded in one fetus of the high-dose group (500 mg/kg bw/d). This finding was not considered biologically relevant.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Some skeletal malformations were detected in all test groups including the control (test groups 0-3; 0, 50, 150 or 500 mg/kg bw/d), as shown in table 5. One control fetus had associated external findings. The incidences of these malformations were neither statistically significantly different from control nor dose-dependent and therefore, not considered biologically relevant.
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dosing. However, the incidence of ‘dumbbell ossification of thoracic centrum (unchanged cartilage)’ was statistically significantly increased in test group 2 (150 mg/kg bw/d). This finding showed no relation to dosing and can be found in the historical control data at a higher frequence. The overall incidences of skeletal variations were also comparable to the historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Soft tissue malformations occurred in one fetus, each, of the control and the high-dose group (0 and 500 mg/kg bw/d). One male high-dose fetus had multiple visceral malformations, i.e. swollen liver lobes (in addition lobular pattern [nutmeg liver]), small kidneys, unexpanded lungs (in addition fused lung lobes), enlarged heart, misshapen and interrupted palatal rugae (in addition to high-arched palate). These visceral findings were associated with an external malformation. The overall incidences of soft tissue malformations were comparable to those found in the historical control data. Details on soft tissue effects are given in table 4.
Five soft tissue variations, i.e. misshapen palatal rugae, short innominate, malpositioned carotid branch, dilated renal pelvis and dilated ureter, were detected. In the majority of cases, the incidences were neither statistically significantly nor dose-dependently changed in comparison to the concurrent control group. However, the incidence of ‘short innominate’ was statistically significantly increased in test group 2 (150 mg/kg bw/d). This finding showed no dose-dependency and was therefore assessed to be without biological relevance. The overall incidences of soft tissue variations were comparable to those found in the historical control data.
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the ribs and the sternum and did not show any relation to dosing.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No adverse effects in fetuses.
Key result
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1: Clinical chemistry (enzymes and substrates + minerals) results of the female dams

Enzymes

 

 

 

 

 

G 0 / F

G 1 /F

50 mg/kgbw/d

G 2 /F

150 mg/kgbw/d

G 3 /F

500 mg/kgbw/d

ALT

Mean

0.98 v

1.10 *

1.33 **

1.83 **

[µkat/L]

S.d.

0.12

0.21

0.17

0.29

day 20

N

25

23

25

25

 

Median

1.01

1.10

1.35

1.79

AST

Mean

1.24 k

1.28

1.22

1.34

[µkat/L]

S.d.

0.32

0.33

0.30

0.46

day 20

N

25

23

25

25

 

Median

1.17

1.23

1.19

1.23

ALP

Mean

1.16 k

1.03

1.02

0.92

[µkat/L]

S.d.

0.31

0.25

0.30

0.26

day 20

N

25

23

25

25

 

Median

1.03

1.03

0.96

0.96

GGT_C

Mean

0

0

0

0

[nkat/L]

S.d.

0

0

0

0

day 20

N

25

23

25

25

 

Median

0

0

0

0

Substrates + minerals

 

 

 

 

 

 

G 0 / F

G 1 /F

50 mg/kgbw/d

G 2 /F

150 mg/kgbw/d

G 3 /F

500 mg/kgbw/d

 

UREA

Mean

4.52 k

4.72

4.58

4.69

 

[mmol/L]

S.d.

0.51

0.86

0.81

0.66

 

day 20

N

25

23

25

25

 

 

Median

4.48

4.53

4.36

4.62

 

CREA

Mean

27.3 k

26.2

27.1

26.8

 

[µmol/L]

S.d.

2.0

2.2

2.2

3.1

 

day 20

N

25

23

25

25

 

 

Median

27.6

26.8

26.8

26.8

 

GLUC

Mean

5.32 k

5.43

5.39

5.45

 

[mmol/L]

S.d.

0.49

0.51

0.41

0.37

 

day 20

N

25

23

25

25

 

 

Median

5.22

5.40

5.32

5.27

 

TBIL_C

Mean

1.26 v

0.98 **

0.94 **

0.81 **

 

[µmol/L]

S.d.

0.25

0.13

0.15

0.22

 

day 20

N

25

23

25

25

 

 

Median

1.23

1.00

0.92

0.75

 

TPROT

Mean

59.59 v

61.63 *

60.90

59.05

 

[g/L]

S.d.

2.06

3.37

3.51

3.88

 

day 20

N

25

23

25

25

 

 

Median

59.76

61.49

60.09

59.04

 

ALB

Mean

35.10 v

36.50 *

36.03

34.73

 

[g/L]

S.d.

1.19

2.34

2.17

2.34

 

day 20

N

25

23

25

25

 

 

Median

34.86

36.21

35.66

35.04

 

GLOB

Mean

24.49 k

25.13

24.88

24.33

 

[g/L]

S.d.

1.11

1.49

1.51

1.73

 

day 20

N

Median

25

24.54

23

24.94

25

24.85

25

24.62

 

CHOL

Mean

2.08 v

2.24

2.41 **

2.53 **

 

[mmol/L]

S.d.

0.32

0.32

0.32

0.41

 

day 20

N

25

23

25

25

 

 

Median

2.03

2.18

2.38

2.50

 

TRIG

Mean

5.28 v

5.31

5.72

6.67 **

 

[mmol/L]

S.d.

1.68

2.23

1.81

1.78

 

day 20

N

25

23

25

25

 

 

Median

5.01

5.45

5.89

6.66

 

INP

Mean

1.62 v

1.71

1.68

1.54

 

[mmol/L]

S.d.

0.22

0.19

0.17

0.20

 

day 20

N

25

23

25

25

 

 

Median

1.55

1.66

1.66

1.55

 

CA

Mean

2.55 v

2.61 **

2.63 **

2.65 **

 

[mmol/L]

S.d.

0.05

0.06

0.09

0.07

 

day 20

N

25

23

25

25

 

 

Median

2.56

2.62

2.63

2.65

 

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

v=KRUSKAL-WALLIS-WILCOX; k=KRUSKAL-WALLIS

Table 2: Absolute and relative organ weights of the female dams

Absolute weights

Females

Test group mg/kg bw/d

1

(50 )

2

( 150)

3

( 500)

Adrenal glands

102%

108%

114%**

Liver

104%

113%**

113%**

Ovaries

101%

108%

112%**

Spleen

103%

108%*

111%*

Relative weights

Females

Test group mg/kg bw/d

1

(50)

2

(150)

3

(500)

Adrenal glands

102%

106%

115%**

Liver

104%

110%**

113%**

Ovaries

101%

106%

112%**

Spleen

103%

106%

111%**

*: p <= 0.05, **: p <= 0.01

Table 3: External examination of the fetuses

Total external observations

 

 

 

 

 

Test group 0

0 mg/kgbw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter Fetuses

N N

25

257

23

210

25

261

25

262

Fetal

incidence

 

N (%)

 

1 (0.4)

 

0.0

 

0.0

 

1 (0.4)

Litter incidence

 

N (%)

 

1 (4.0)

 

0.0

 

0.0

 

1 (4.0)

Affected

fetuses/litter

 

Mean%

 

0.4

 

0.0

 

0.0

 

0.4

Total external unclassified observations

 

 

 

 

 

Test group 0

0 mg/kgbw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter Fetuses

N N

25

257

23

210

25

261

25

262

Fetal incidence

 

N (%)

 

0.0

 

0.0

 

0.0

 

1 (0.4)

Litter incidence

 

N (%)

 

0.0

 

0.0

 

0.0

 

1 (4.0)

Affected fetuses/litter

 

Mean%

 

0.0

 

0.0

 

0.0

 

0.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 4: Soft tissue examination of the fetuses

Total soft tissue malformations

 

 

 

 

 

Test group 0

0 mg/kgbw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter Fetuses

N N

25

121

22

99

25

125

25

124

Fetal incidence

 

N (%)

 

1 (0.8)

 

0.0

 

0.0

 

1 (0.8)

Litter incidence

 

N (%)

 

1 (4.0)

 

0.0

 

0.0

 

1 (4.0)

Affected fetuses/litter

 

Mean%

 

0.8

 

0.0

 

0.0

 

0.8

Total soft tissue variations

 

 

 

 

 

Test group 0

0 mg/kgbw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter Fetuses

N N

25

121

22

99

25

125

25

124

Fetal incidence

 

N (%)

 

6 (5.0)

 

2 (2.0)

 

5 (4.0)

 

7 (5.6)

Litter incidence

 

N (%)

 

5 (20)

 

2 (9.1)

 

4 (16)

 

5 (20)

Affected fetuses/litter

 

Mean%

 

4.7

 

5.5

 

3.9

 

5.7

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 5: Skeletal examination of the fetuses

Total skeletal malformations

 

 

 

 

Test group 0

0 mg/kgbw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter Fetuses

N N

25

136

23

111

25

136

25

138

Fetal

incidence

 

N (%)

 

1 (0.7)

 

1 (0.9)

 

0.0

 

1 (0.7)

Litter incidence

 

N (%)

 

1 (4.0)

 

1 (4.3)

 

0.0

 

1 (4.0)

Affected

fetuses/litter

 

Mean%

 

0.6

 

1.1

 

0.0

 

1.0

Total fetal skeletal variations

 

 

 

 

Test group 0

0 mg/kgbw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter Fetuses

N N

25

136

23

111

25

136

25

138

Fetal incidence

 

N (%)

 

133 (98)

 

108 (97)

 

132 (97)

 

132 (96)

Litter incidence

 

N (%)

 

25 (100)

 

23 (100)

 

25 (100)

 

25 (100)

Affected fetuses/litter

 

Mean%

 

97.7

 

97.2

 

96.9

 

95.4

Total unclassified cartilage observations

 

 

 

 

Test group 0

0 mg/kgbw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter Fetuses

N N

25

136

23

111

25

136

25

138

Fetal incidence

 

N (%)

 

119 (88)

 

94 (85)

 

109 (80)

 

110 (80)

Litter incidence

 

N (%)

 

25 (100)

 

22 (96)

 

23 (92)

 

24 (96)

Affected fetuses/litter

 

Mean%

 

88.2

 

80.0

 

78.3

 

80.1

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Conclusions:
The administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of maternal toxicity, such as an anemia and altered liver cell metabolism.
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 150 mg/kg bw/d.
There were no toxicologically relevant adverse fetal findings evident. Thus, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 500 mg/kg bw/d.
The test substance is not teratogenic in rats.
Executive summary:

SUMMARY

METHODS

Tris-(2-ethylhexyl)amine was tested for its prenatal developmental toxicity in Wistar rats.

The test substance was administered as an oily solution to groups of 25 time-mated female Wistar rats by gavage at doses of 50, 150 and 500 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (corn oil) in parallel. A standard dose volume of 4 mL/kg body weight was used for each test group.

At terminal sacrifice on GD 20, 23-25 females per group had implantation sites.

OBSERVATIONS

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the adrenal glands, kidneys, liver, ovaries, spleen, unopened uterus and placentas).

For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

RESULTS

Analytics

The various analyses confirmed

• the stability of the test substance preparations over a period of 7 days at room temperature,

• the correctness of the prepared concentrations.

Effects

The following test substance-related adverse effects/findings were noted:

Test group 3 - 500 mg/kg bw/d:

Dams

• Decreased red blood cell (RBC) counts, hemoglobin and hematocrit values

• Increased alanine aminotransferase (ALT) activities

• Increased cholesterol and triglyceride values

• Increased absolute and relative organ weights of liver and spleen

Fetuses

• No test substance-related adverse effects

Test group 2 - 150 mg/kg bw/d:

• No test substance-related adverse effects

Test group 1 - 50 mg/kg bw/d:

• No test substance-related adverse effects

CONCLUSION

Under the conditions of this prenatal developmental toxicity study, the oral administration of Tris-(2-ethylhexyl)amine to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of maternal toxicity, such as anemia and altered liver cell metabolism.

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 150 mg/kg bw/d.

There were no toxicologically relevant adverse fetal findings evident.

Thus, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 500 mg/kg bw/d.

The test substance is not teratogenic in rats.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The test substance was tested for its prenatal developmental toxicity in Wistar rats (OECD 414). The test substance was administered as an oily solution to groups of 25 time-mated female Wistar rats by gavage at doses of 50, 150 and 500 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (corn oil) in parallel.

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the adrenal glands, kidneys, liver, ovaries, spleen, unopened uterus and placentas).

For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) finding

The following test substance-related adverse effects/findings were noted for the dams of test group 3 (500 mg/kg bw/d); Decreased red blood cell (RBC) counts, hemoglobin and hematocrit values, increased alanine aminotransferase (ALT) activities, increased cholesterol and triglyceride values, increased absolute and relative organ weights of liver and spleen. The fetuses showed no test substance-related adverse effects. For the other test groups (150 and 50 mg/kg bw/d) no test substance-related adverse effects were noted.

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of maternal toxicity, such as anemia and altered liver cell metabolism.

The no observed adverse effect level (NOAEL) for maternal toxicity is 150 mg/kg bw/d. There were no toxicologically relevant adverse fetal findings evident. Thus, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 500 mg/kg bw/d. The test substance is not teratogenic in rats.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008, as amended for the thirteenth time in Regulation (EU) No 2018/1480. As a result classification of the substance for toxicity to reproduction is warranted Cat. 2 (H361 f "Suspected of damaging fertility"). Mating behaviour, conception and parturition were not effected in an OECD 422 guideline study, however, the slightly lower number of implants in all treated groups was suggested to be a potential adverse effect on reproductive performance.

Additional information