bis(hydrogenated tallow C16-18-alkyl)hydroxylamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a 1-generation study treatment with the test item by oral gavage in male and female Wistar rats at dose levels of 10, 50 and 200 mg/kg body weight/day revealed no parental, reproduction, breeding and developmental toxicity for treatment up to 200 mg/kg body weight/day. Based on these results, the definitive parental, reproduction, breeding and developmental No Observed Adverse Effect Level (NOAEL) and No Observed Effect Level (NOEL) was established as being 200 mg/kg body weight/day.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07.09. - 19.01.2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Version / remarks:

adopted 1983

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Males: Charles River Deutschland, Sulzfeld, Germany.
Females: Charles River Laboratories, Wilmington, MA, USA.
- Age at study initiation: Males 5-6 weeks and females 13-14 weeks.
- Housing:
Pre-mating animals were housed in groups of 4 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating Females were caged together with males on a one-to-one-basis in Macrolon cages (M III type, height 18 cm).
Post-mating Males were housed in groups of 4 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). Females were individually housed in Macrolon cages (M III type, height 18 cm).
Lactation Offspring was kept with the dam until termination.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days (males) or 4 days (females) prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): temperature of 21 ± 3°C (measured range: 19 -23.4°C)
- Humidity: 30 - 70 % (actual range: 35 - 100 %)
- Air changes (per hr): 15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES:
Males: from 06.09. to 18. and 28.12.2006
Females: from 01.11.2007 to 03.-19.2007

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % (w/w) carboxymethylcellulose and 0.1% (v/v) Tween-80 in Milli-U water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

From start study to 14 November 2006, formulations (w/w) were prepared weekly and were homogenised to a visually acceptable level. From 15 November 2006 to end of the study, formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. No adjustment was made for specific gravity of the test substance, vehicle, and/or formulation.

VEHICLE
- Justification for use and choice of vehicle:appropriateness of vehicle was shown in a 90 d oral gavage study in rats
- Concentration in vehicle: 40, 10 or 2 mg/mL
- Amount of vehicle: 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: one female was cohabitated with one male of the same treatment group, avoiding sibling mating
- Length of cohabitation: 15 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of a intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility for an additional 5 days.
- Further matings after two unsuccessful attempts: no
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Remarks:

The concentrations in the formulation of Group 4 were in agreement with target concentrations and homogenous. As no toxicity was observed up to and including the high dose group, the lower accuracies for group 3 and 2 did not affect the study integrity.

Details on analytical verification of doses or concentrations:

Accuracy, homogeneity and stability were determined for formulations prepared for use in Week 1, Week 8, Week 16 and Week 18 of study.
The concentrations analysed in the formulations of Group 4 (200 mg/kg bw/d) were in general in agreement with target concentrations (i.e. between 90% and 110%). The analysed concentrations of the formulations of Group 3 (50 mg/kg bw/d) were somewhat lower than the target concentrations. The formulations of Group 4 were homogeneous. Formulations were stable for at least 5 hours when stored at room temperature.
For formulations of Group 2 (10 mg/kg bw/d), relatively low accuracies and high coefficients of variation on the accuracies were obtained. As no toxicity was observed up to and including the high dose group, the relatively low accuracies and high coefficients of variation on the accuracies observed for the low dose formulations were not considered to have affected the study integrity.

Duration of treatment / exposure:

F0-males: A minimum of 70 days prior to mating and continuing until necropsy.
F0-females: A minimum of 14 days prior to mating and continuing until necropsy.
F1-generation (F0-offspring): The F1-generation was potentially exposed to the test substance in utero and through nursing during lactation.

Frequency of treatment:

Once daily for 7 days per week. Animals were dosed up to the day prior to scheduled necropsy.

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were derived from a 90-day oral toxicity study in rats with a NOAEL of 50 mg/kg bw and effects on liver, kidney and mesenteric lymph nodes at doses of 200 and 1000 mg/kg bw/d.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
At least once daily, detailed clinical observations were made in all animals. The time of onset, degree and duration were recorded.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among the pregnant females.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 of gestation, and during lactation on Days 1, 4, 7,14 and 21.

FOOD CONSUMPTION:
Weekly, for males and females. Food consumption was not recorded during the breeding period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and after delivery on Days 1, 4, 7, 14 and 21 post-partum.

WATER CONSUMPTION:
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OTHER:
REPRODUCTION PROCESSES: Male number paired with, mating date, confirmation of pregnancy, and delivery date were recorded.

Oestrous cyclicity (parental animals):

Not determined.

Sperm parameters (parental animals):

Parameters examined in all male parental generations: testis weight, epididymis weight, no sperm parameters examined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
To reduce variability among the litters, on Day 4 of lactation, eight pups from each litter of equal sex distribution (if possible) were randomly selected. For litters consisting of fewer than eight pups, adjustments for litter size were not performed.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
Mortality/ viability
The numbers of live and dead pups at the First Litter Check (=check at day 1 of lactation) and daily thereafter were determined.

Clinical signs
At least once daily, detailed clinical observations were made in all animals.

Body weigts
Live pups were weighed during lactation on days 1, 4, 7, 14 and 21.

Sex
Was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

Postmortem examinations (parental animals):

SACRIFICE
All animals surviving to the end of the observation period and all moribund animals were anaesthetised using iso-flurane and subsequently exsanguinated. Males were killed as soon as possible after delivery of the litters. Females were killed on Day 21 postpartum or shortly thereafter.

GROSS NECROPSY
After sacrifice or death all animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10 % buffered formalin: Cervix, Coagulation gland, kidneys, liver, meseneteric lymph nodes, epididymides, ovaries, pituitary gland, prostate gland, seminal vesicles, testes, uterus, vagina, all gross lesions.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following slides were examined by a pathologist:
The kidneys, liver, mesenteric lymph nodes of 10 mated animals/sex of Group 1 and Group 4.
On detection of possible treatment-related changes in the mesenteric lymph nodes of high dose males, histological examination was extended to that particular organ of 10 mated males of Group 2 and Group 3.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy:
Epididymides, kidneys, liver, ovaries, pituitary (weighed when fixed for at least 24 hours), prostate (weighed when fixed for at least 24 hours), seminal vesicles, testes, uterus.

Postmortem examinations (offspring):

SACRIFICE
Pups, which were culled, were killed by decapitation. All remaining pups were sacrificed using an oxygen/carbon dioxide procedure.
Culling was performed on Day 4 of lactation or shortly thereafter. The remaining pups were killed at Day 21 post partum or shortly thereafter.

GROSS NECROPSY
Offspring found dead or killed before Day 14 of lactation was sexed and externally examined (if practically possible) with emphasis on developmental morphology. The stomach was examined for the presence of milk.
Offspring killed on or after Day 14 of lactation was sexed and subjected to external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs with emphasis on developmental morphology. Descriptions of all macroscopic abnormalities were recorded. If possible, defects or cause of death were evaluated. No pups or tissues were fixed.

Statistics:

The following statistical methods were used to analyze the data:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate, was applied for the comparison of the treated groups and the control groups for each sex.
The Steel-test (Miller, 1981 ) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
The Fisher Exact-test (Fisher 1950) was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Reproductive indices:

Percentage mating, fertility index, conception rate, gestation index, gestation duration.

Offspring viability indices:

Percentage live males at first litter check, percentage live females at first litter check, percentage of postnatal loss days 0-4 post partum, percentage of breeding loss day 5 until weaning, percentage live males at weaning, percentage live females at weaning, viability index, weaning index.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment related clinical signs were noted. The female of the high dose group (animal number 188) that was killed in extremis showed hunched posture for one day and piloerecfion and pale appearance for two days. Incidental findings consisted of chromodacryorrhoea of the snout and pale appearance. In addition, animals of all groups showed alopecia and/or scabs. These findings are occasionally noted in rats of this age and strain that are housed and handled under the conditions in this study and are therefore considered to be of no toxicological significance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

At the high dose group, one female was killed in extremis on Day 42 of treatment. This female showed hunched posture, piloerection and pale appearance. Macroscopic examination revealed an enlarged spleen. In addition, four implantation sites were observed in the left uterus horn, ten mummified foetuses were observed in the right uterine horn and two mummified foetuses were noted in the cervix . This death was not regarded as treatment-related but considered related to delivery difficulties. No further unscheduled deaths occurred.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the complete study period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption before or after allowance for body weight was similar between treated and control animals. The statistically significantly increased value for relative food consumpfion observed for females of the intermediate dose group during the lactafion period was considered unrelated to treatment as no dose response relafionship was apparent and this finding was very slight and incidental.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related microscopic findings were observed.
Microscopic findings were present in the mesenteric lymph nodes of males. Macrophage foci were present in all groups: In Group 1 (5/10 males: minimal). Group 2 (7/10 males: minimal). Group 3 (4/10 males: minimal, 1/10 male: slight) and Group 4 (5/10 males: minimal, 2/10 males: slight). The findings in the mesenteric lymph nodes and the remainder of the microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar rats of this age and strain.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproduction parameters were unaffected by treatment up to 200 mg/kg bw/d.

Key result

Dose descriptor:

NOAEL

Effect level:

> 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item-related effects up to and incl. the higher dose level of 200 mg/kg bw/d.

Critical effects observed:

not specified

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental clinical symptoms consisted of no milk, small and pale appearance, opacity of the eye, ptosis and watery discharge from eye (both not confirmed at macroscopic examination). In addition, pups showed alopecia, scabs, fissures in the axillary region, wound, blue abdomen, brown wrinkled tail, missing tail apex (not confirmed at macroscopic examination) and/or thickened (red-blue) area of the head. These findings are occasionally noted in rats of this age and strain that are housed and handled under the conditions in this study and are therefore considered to be of no toxicological significance.

Mortality / viability:

no mortality observed

Description (incidence and severity):

Breeding parameters were unaffected by treatment up to 200 mg/kg bw/d. Postnatal loss, living pups on Day 4 post partum, breeding loss between Days 5-21 post partum, living pups on Day 21 post partum, viability index and weaning index were similar for the control and treated groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of treated groups remained in the same range as controls.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic examination of the pups revealed small appearance, no milk, tail apex damaged and black discoloured (correlating to the brown wrinkled tail), pelvic dilation of the kidney, watery cyst at the left kidney and dilated left ureter, wound, hernia diaphragmatica liver, and grey-white discolourafion of the right eye (correlating to opacity of the eye). No relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.

Histopathological findings:

not examined

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item-related effects up to and incl. the higher dose level of 200 mg/kg bw/d.

Critical effects observed:

not specified

Key result

Reproductive effects observed:

no

REPRODUCTION DATA

Number of females	Group 1 (control)	Group 2 (10 mg/kg)	Group 3 (50 mg/kg)	Group 4 (200 mg/kg)
Paired	24	24	24	24
Mated	24	23	23	24
Pregnant	24	23	23	24
Dams killed in extremis	0	0	0	1
Females with living pups	24	23	23	23

FERTILITY F0-GENERATION

	Group 1 (control)	Group 2 (10 mg/kg)	Group 3 (50 mg/kg)	Group 4 (200 mg/kg)
Percentage mating	100	95.8	95.8	100
Fertility index	100	95.8	95.8	100
Conception rate	100	100	100	100
Gestation index	100	100	100	95.8

Conclusions:

Based on these results, the definitive parental, reproduction, breeding and developmental No Observed Adverse Effect Level (NOAEL) and No Observed Effect Level (NOEL) was established as being 200 mg/kg body weight/day.

Executive summary:

In this one-generation study, the test item was administered by oral gavage through one complete reproductive cycle to SPF-bred male and female Wistar rats. The dose levels tested were 0,10, 50 and 200 mg/kg body weight/day. The dose levels were derived from a 90-day oral toxicity study in Ralf (SPS) rats with a NOAEL of 50 mg/kg bw and effects on liver, kidney and mesenteric lymph nodes at doses of 200 and 1000 mg/kg body weight/day. There were no changes for mortality, clinical signs, body weight, food consumpfion, macroscopic examination, organ weights, reproduction, breeding data and pup development that were considered to be an effect of treatment. In the mesenteric lymph nodes of males, macrophage foci were slighty increased in the higher dosed animals. This was considered to be within the normal range for Wistar rats of this age and strain. Treatment with the test item by oral gavage in male and female Wistar rats at dose levels of 10, 50 and 200 mg/kg body weight/day revealed no parental, reproduction, breeding and developmental toxicity for treatment up to 200 mg/kg body weight/day. Based on these results, the definitive parental, reproduction, breeding and developmental No Observed Adverse Effect Level (NOAEL) and No Observed Effect Level (NOEL) was established as being 200 mg/kg body weight/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subchronic

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

A one-generation reproduction toxicity study following OECD guideline 415 was performed with the test material in rats by oral gavage. After acclimatization, male and female Wistar rats of the F0 generation were exposed by oral gavage to graduated doses of the test substance. The dose levels were 10, 50 and 200 mg/kg body weight/day. The dose levels were derived from a 90-day oral toxicity study in Ralf (SPS) rats with a NOAEL of 50 mg/kg bw and effects on liver, kidney and mesenteric lymph nodes at doses of 200 and 1000 mg/kg body weight/ day. Chemical analyses of formulations were conducted during the study to assess accuracy, homogeneity and stability. Males were exposed 10 weeks and females 2 weeks prior to mating and exposure ended at termination. F0 females were allowed to produce and rear a litter until Day 21 of lactation. On Day 4 of lactation litters were reduced in size to eight pups by random culling of F1 pups. All animals were subjected to daily clinical observation. Body weight and food consumption were measured on several occasions over the treatment period. At necropsy, macroscopic observations and organ weights were recorded. Reproduction parameters, breeding data and pup development were assessed. Histopathology of liver, kidney and mesenteric lymph nodes was performed because these organs were identified as target organs in a subchronic toxicity study. There were no changes for mortality, clinical signs, body weight, food consumption, macroscopic examination, organ weights, reproduction, breeding data and pup development that were considered to be an effect of treatment. In the mesenteric lymph nodes of males, macrophage foci were slightly increased in the higher dosed animals. This was considered to be within the normal range for Wistar rats of this age and strain. From the results presented in this report a definitive parental, reproduction, breeding and developmental No Observed Adverse Effect Level (NOAEL) and No Observed Effect Level (NOEL) for the test substance of 200 mg/kg/day was established.

Effects on developmental toxicity

Description of key information

No developmental toxicity / teratogenicity was evaluated. However, the One-generation study did not indicate any effect on the F1 generation that may be suspicious for a disturbance of the development of the offspring.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the data available the substance is not considered to be classified for toxicity to reproduction under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218.