2-[(2-[2-(dimethylamino)ethoxy]ethyl)methylamino]ethanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a key, K1 one-generation reproductive toxicity study (Martell, 2013), the test substance was administered orally to male and female rats at dose levels 10, 50 and 100 mg/kg bw/day (OECD guideline 415). The NOAEL was established to be greater than 100 mg/kg bw/day for males and females systemic and reproductive toxicity.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-06 to 2013-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Experimental test run under GLP conditions and according to OECD Guidelines with no deviations identified. Study conducted in an AAALAC – Accredited Test Facility in conformity with the applicable rules for animal welfare and humane use and care of laboratory animals.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Test substance: JEFFCAT ZF-10
Chemical name (IUPAC): N,N,N'-Trimethyl-N'-hydroxyethyl-bis-aminoethyl ether
CAS number: 83016-70-0
BIOAGRI code: AGR-1892/11
Sample arrival date: 28/Dec/2011
Batch number: IK703
Expiration date: 18/Jun/2013
Storage: stored in a specific room (Test Item Storage Room), at room temperature, protected from humidity and light

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
a) Species: rat (Rattus norvegicus)
b) Strain: Wistar Hannover
c) Source: BIOAGRI Laboratórios – DF’s breeding house
d) Sex: males and females (nulliparous and non-pregnant)
e) Age at starting of dosing: 7 to 8 weeks old
f) Number of animals: 13 males/group and 26 females/group.

- Weight at study initiation: no details
- Fasting period before study: yes
- Housing: polypropylene cages (41x34x19 cm) with wire mesh tops and bedding material (wood shavings)
- Diet (e.g. ad libitum): Nuvilab CR-1 diet for rats supplied by Nuvital Nutrientes Ltda. available ad libitum
- Water (e.g. ad libitum): filtered drinking water was autoclaved. Water was supplied by CAESB (Companhia de Saneamento Ambiental do Distrito
Federal)
- Acclimation period: 3 males/cage and up to 2 females/cage examined and acclimated for minimum 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.6 – 24°C
- Humidity (%): 42.5 – 69.6%
- Air changes (per hr): 10-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized

Details on exposure:

Dosing began 70 days before mating for males and 14 days for females and continued until the day prior to necropsy (after twenty-one days of
lactation for females and at the end of the mating period for males).

Details on mating procedure:

Premating :
At the end of acclimation period, animals were randomly assigned to the experimental groups, housed (up to 3 males/cage and up to 2 females/cage) and the treatment began.
Mating :
After a premating period of 70 days for males and 14 days for females, two females were cohabited with an assigned male (2 female: 1 male) from the same dose level until evidence of copulation was observed, or 3 weeks had elapsed. Care was taken to avoid sibling mating. Vaginal smears were collected daily during mating period and examined for the presence of sperm. Day 0 of gestation was defined as the day a sperm is found in the vaginal smear. Female No 10 (control group) that showed no evidence of copulation at the end of the mating period (3 weeks) was placed with a second male at the same group and mating was confirmed 2 days later.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Parental animals (males) were treated 70 days before mating and until the end of the mating period.
Male animals were dosed for a total of 94 days.
For females, this included 14 days prior to mating, during the mating period (up to 4 weeks), during gestation and up to lactation day 20, for a total of 41 to 80 days.

Frequency of treatment:

Daily, by gavage, on a 7-day-week-basis.
The volume administered each day was 4 mL/kg body weight.

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

In deionized water

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

In deionized water

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

In deionized water

No. of animals per sex per dose:

3 groups of 13 male and 26 female rats per dose
Control group of 13 males and 26 females

Control animals:

yes, concurrent vehicle

Details on study design:

- dose level selection: the dosage levels were selected following consultation with the Sponsor on the basis of the results of previous testing with the test substance. The following doses were chosen for this study:
* 10 mg/kg bw/day as the expected dose which causes no signs
* 50 mg/kg bw/day as the intermediate dose level
* 100 mg/kg bw/day as the expected dose which causes signs of systemic toxicity, but not death or severe suffering

Test group Dose Concentration Number of animals Animal identification
(mg/Kg/day) (mg/mL) Male Female Male Female
1 Vehicle (a) 0 13 26 105-117 1-26
2 10 (b) 2,5 13 26 118-130 27-52
3 50 (b) 12,5 13 26 131-143 53-78
4 100 (b) 25 13 26 144-156 79-104

(a) Vehicle (deionized water).
(b) test substance (Test Solutions).

Parental animals: Observations and examinations:

Throughout the treatment period, each animal was observed at least once daily for mortality, morbidity, pertinent behavioral changes, signs of difficult or prolonged parturition, and all signs of overt toxicity through cage side observations. At least once weekly throughout the treatment period (except during mating and gestation), each animal was handled and examined for signs of ill health or reaction to treatment, abnormal behavior or appearance.

Litter observations:

Litters were weighed on days 0 and 4 (before standardization) and after standardization on days 7 and 14. Live pups were weighed individually after standardization on day 21.

Postmortem examinations (parental animals):

All animals were euthanized by CO2 inhalation:
- Males: until the end of mating period;
- Females: from day 21 post-partum;
- Females which have not delivered at day 25 post-coitum: From day 25 post-coitum.
At termination, all parental animals were examined macroscopically for any abnormalities or pathological changes, with special attention paid to the organs of the reproductive system.

Postmortem examinations (offspring):

All animals were euthanized by CO2 inhalation:
- Surviving pups: on day 4 of lactation which were eliminated after standardization: From day 4 post-partum;
- Surviving pups: from day 21 post-partum.
Dead pups and pups euthanized at day 4 were necropsied for macroscopic evaluation, preserved and studied for possible defects.

Statistics:

Quantitative variables such as body weights, food consumption, number of fetuses and corpora lutea were analyzed by One Way Analysis of Variance (ANOVA), followed by Dunnett’s test if significance was detected, or by non-parametric test of Kruskal-Wallis, according to the results of tests for normality and homogeneity of variance. The Chi-Square Test was used for statistical evaluation of clinical findings, macroscopic and microscopic findings and loss of offspring. The level of significance was set at 5%, and the statistical program used was SAS Software (SAS Institute Inc., Cary, NC).

Clinical signs:

no effects observed

Description (incidence and severity):

Test substance did not cause clinical signs.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

Test substance did not cause mortality.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effects on body weight, body weight gain were observed.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No effect on food consumption was observed.

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:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no microscopic alterations that could be attributed to the test item.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The male and female mating indices were 100% in all groups, as were the male fertility index and the gestation index. However, the female fertility index was lower than male, although it was similar across all groups.

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

> 100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

> 100 mg/kg bw/day

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed in pups through day 21 postnatal.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No test item related pup mortality occurred.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Statistically significant lower mean body weight in pups from dams exposed to 10 mg/kg/day. difference was very slight in magnitude and occurred in the low dose level, it was considered to be incidental.

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

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

The necropsy evaluation did not reveal treatment related findings in pups.

Histopathological findings:

not examined

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Physical evaluation of male and female pups did not reveal any effects of test item administration in the different groups. This includes the statistically significant delayed day of pinna detachment appearance in females at 100 mg/kg/day (+5.2%) when compared to the control group.
Despite that this finding was observed at the high dose level, it was not considered to be treatment related because was an isolated finding and occurred in very slight magnitude.
In the reflex evaluation of the pups, no important alteration occurred in treated males or females compared to the control group. Only numerical differences were found and considered as normal biological variations, even in the statistically significant observations which were not dose
proportional.

Key result

Dose descriptor:

NOAEL

Remarks:

embryofetal toxicity

Generation:

F1

Effect level:

> 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Test substance did not cause mortality or clinical signs of toxicity during the study. No effects on body weight, body weight gain or food consumption were observed. No treatment related macroscopic changes were noted in male or female rats. There were no microscopic alterations that could be attributed to the test item. None of the mating, gestation or lactation parameters were considered to have been affected by treatment with the test item. No biologically significant differences were found between the numbers of live or dead pups from treated and control dams during whole treatment period. No clinical signs were observed in pups through day 21 postnatal. No test item related pup mortality occurred. The mean body weight of pups on days 0, 4, 7, 14 and 21 postnatal were similar in all groups. Physical and reflex evaluation of male and female pups did not reveal effects of test item administration in the different groups. The necropsy evaluation did not reveal treatment related findings in pups.

Conclusions:

In the experimental conditions of this one generation reproduction toxicity study, the No Observed Adverse Effect Level (NOAEL) of the test item test substance in Wistar rats was considered to be greater than 100 mg/kg/day for males and females and greater than 100 mg/kg/day for maternal-embryo-fetal toxicity.

This test was requested by the National Competent Authority in accordance with Article 16(1) of Directive 67/548/EEC.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 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 reproductive toxicity study was performed for the test substance according to OECD Guideline 415 (Martell, 2013). The test substance was administered to male and female animals in dose levels 10, 50 and 100 mg/kg bw/day for a total of 94 days for males (70 days before mating and until the end of mating period) and for a total of 41 to 80 days for females (14 days prior to mating, during the mating period (up to 4 weeks), during gestation and up to lactation day 20.

During the dosing period, animals were observed twice daily for mortality and clinical signs on working days and once a day. Males were weighed on the first day of dosing and weekly thereafter. Females were weighed on the first day of dosing, once weekly during the premating and mating periods, on days 0, 7, 14 and 20 of gestation and on days 0, 4, 7, 14 and 21 of lactation. Food consumption was recorded on the same days as the body weight determinations, except on day 0. Females of all groups were allowed to deliver normally and to rear their progeny until day 21 postpartum. Pregnancy and litter parameters were recorded, and during the lactation period each pup was observed daily for mortality and clinical signs. Physical development such as eye opening, incisor eruption, unfolding of external ear and behavioral abnormalities observed were recorded. Functional investigations (reflex ontogeny) were carried out on the pups during the lactation period. On day 4 of lactation, the size of each litter was adjusted by eliminating extra pups by random selection to yield 4 males and 4 females per litter. Dead pups and pups killed at day 4 were preserved and studied for possible defects. Pup body weight was recorded on days 0, 4 and 7 and weekly thereafter until termination of the study, when animals were weighed individually. A macroscopic post-mortem examination was performed on all parental animals and all macroscopic lesions were collected and preserved. Histopathology of vagina, uterus with cervix, ovaries, testes, epididymides, seminal vesicles and coagulating gland was performed in high dose and control animals. Histopathology of prostate, pituitary gland, thyroid, adrenal, liver, kidney, lung, heart and spleen was performed in 5 animals/sex/group for the control and high dose group. The reproductive organs of animals No 36, 43 and 49 (females) at the low dose and female No 56 at the mid dose, suspected of infertility were subjected to microscopic examination. All gross lesions found were examined.

Physical evaluation of male and female pups did not reveal any effects of test item administration in the different groups. This includes the statistically significant delayed day of pinna detachment appearance in females at 100 mg/kg/day (+5.2%) when compared to the control group. Despite that this finding was observed at the high dose level, it was not considered to be treatment related because was an isolated finding and occurred in very slight magnitude.

In the reflex evaluation of the pups, no important alteration occurred in treated males or females compared to the control group. Only numerical differences were found and considered as normal biological variations, even in the statistically significant observations which were not dose proportional. The test substance did not cause mortality or clinical signs of toxicity during the study. No effects on body weight, body weight gain or food consumption were observed. No treatment related macroscopic changes were noted in male or female rats. There were no microscopic alterations that could be attributed to the test item. None of the mating, gestation or lactation parameters were considered to have been affected by treatment with the test item. No biologically significant differences were found between the numbers of live or dead pups from treated and control dams during whole treatment period. No clinical signs were observed in pups through day 21 postnatal. No test item related pup mortality occurred. The mean body weight of pups on days 0, 4, 7, 14 and 21 postnatal were similar in all groups. Physical and reflex evaluation of male and female pups did not reveal effects of test item administration in the different groups. The necropsy evaluation did not reveal treatment related findings in pups. In the experimental conditions of this one generation reproduction toxicity study, the NOAEL of the test substance in Wistar rats was considered to be greater than 100 mg/kg/day for males and females and greater than 100 mg/kg/day for maternal-embryo-fetal toxicity.

Effects on developmental toxicity

Description of key information

A read-across prenatal development test with the structural analogue substance N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine) in male/female New Zealand White rabbits was performed according to OECD Guideline 414. Based on the findings, the maternal toxicity NOAEL was between 1 and 5% (or 2.4 -12 mg/kg bw/day). The maternal toxicity consisted of transient reduced weight gain, renal lesions, and elevated relative kidney weight. The fetal toxicity was between 5 and 10% (12-24 mg/kg bw/day). The toxicity to the fetus is based on a reduced fetal body weight per litter without any indication of reduced fetal ossification.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non GLP, and animals were only treated from gestation days 6-18. However, study was well documented, sufficient number of animals and doses were employed, and a vehicle and untreated control group were included.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

treatment period limited to gestation day 6-18

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): NIAX Catalyst A-99, (bis(2-dimethylaminoethyl)ether)
- Physical state: transparent, yellowish, non-viscous liquid
- Analytical purity: 98.7 %
- Lot/batch No.: 897066
- Impurities (identity and concentrations): 0.723 % 2(2-dimethylaminoethoxy) ethanol, and the rest water

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Dutchland Laboratories, Inc., Denver, PA
- Age at study initiation: 5-5.5 months
- Weight at study initiation: 3-3.5 kg
- Housing: Animals were individually housed in stainless steel wire-mesh cages (46 cm x 61 cm x 36 cm high)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 2 weeks


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: September 22, 1984 To: October 19, 1984

Route of administration:

dermal

Vehicle:

other: distilled water

Details on exposure:

TEST SITE
- Area of exposure: a 3x3 inch area on the mid dorsum
- Type of wrap if used: After application a 3x3 inch, 12-ply, sterile gauze was applied over the dosing site. The gauze was held in place by two strips (approximately 8 inches long) of 3-inch wide, elastic adhesive tape.
- Time intervals for shavings or clipplings: A 3x3 inch area on the mid dorsum of all dosed female rabbits was clipped and shaved one to two days prior to treatment initiation (gestation day 4 or 5) and any time throughout treatment as deemed necessary due to rapid hair growth.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The application site was wiped gently with a gauze strip dampened with warm water and blotted dry with a disposable wiper.
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1 ml of test substance in vehicle
- Concentration (if solution): 1, 5 or 10 %
- Constant volume or concentration used: yes

VEHICLE
- Amount(s) applied (volume or weight with unit): 1 ml

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the dosing formulations for stability and test substance content (pre-and post-doing) were provided by Dr. R.A. Budnik, Union Carbide Corporation, Research and Development, Silicones and Urethane Intermediates Division, Tarrytown, NY. The weight % test substance values were determined by potentiometric titration. The volume % values were calculated from the weight % using a density of 0.8492 g/cc for the test substance.
The analysis of dosing formulations prepared on September 10, 1984 indicated that all formulations were within 99.8-104.0 % of target prior to dosing period. According to the Sponsor, aqueous formulations were stable for at least several months in stoppered containers at ambient temperate. The post dosing analysis indicated the formulations were within 93-96.2 % of target.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: Rabbits were mated consecutively to two males (if possible). After the second copulation, females were individually housed.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: The date of copulation(s) was designated gestation day 0.

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

Animals were exposed daily on gestation days 6-18.

Duration of test:

Females were sacrificed on gestation day 29.

Dose / conc.:

1 other: %

Remarks:

2.4 mg/kg (average for dose group)

Dose / conc.:

5 other: %

Remarks:

12 mg/kg (average for dose group)

Dose / conc.:

10 other: %

Remarks:

24 mg/kg (average for dose group)

No. of animals per sex per dose:

22

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were based on results from a dose range-finding study also performed on timed-pregnant New Zealand White rabbits.
- Rationale for animal assignment: Mated females were assigned by stratified randomization by body weight to each experimental group.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included. All females were examined for clinical signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The treated does were observed daily for skin irritation, erythema, eschar formation and edema.

BODY WEIGHT: Yes
- Time schedule for examinations: All females were weighed on gestation day 0, 6 (prior to onset of dosing), 12, 18 (during the dosing period), and 29.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: the gravid uterus, ovaries (including corpora lutea), cervix, vagina, and peritoneal and thoracic cavities were examined grossly. Maternal liver, kidneys and uterine weights were determined. Kidneys were bisected, fixed in buffered neutral 10 %formalin and processed for histopathological examination. One square inch of skin from treated animals was removed from the application site and fixed in buffered neutral 10 % formalin. The uteri were immediately ligated at their cervical end to prevent the expulsion of conceptuses by myometrial peristalsis. Uteri were externally examined for signs of hemorrhage. The ligated uteri and attached ovaries and oviducts were removed from the peritoneal cavity and weighed.

Ovaries and uterine content:

The ovaries and uterine content were 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
- Other: number of live and dead fetuses

Fetal examinations:

- External examinations: Yes, all live fetuses per litter
- Soft tissue examinations: Yes, approx. half per litter
- Skeletal examinations: Yes, remaining fetuses not used for soft tissue examinations

Statistics:

The unit of comparison was the pregnant female or the litter. Results of the quantitative continuous variables (e.g., maternal body weights, liver weights, fetal weights, etc.) were intercompared for the test substance groups, the untreated control group and vehicle control group by use of Levene's test for equal variances, analysis of variance (ANOVA), and t-tests with Bonferroni probabilities. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated homogenous variances and the ANOVA was significant, the pooled t-test was used. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by the separate variance t-test.
Non parametric data obtained following laparohysterectomy were statistically treated using the Kruskal-Wallis test followed by the Mann-Whitney U test when appropriate. Incidence data were compared using Fisher's Exact Test. For all statistical tests, the fiducial limit of 0.05 (two-tailed) was used as the criterion for significance.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs, not related to dermal irritation, appeared to be treatment-related.

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

The clinical observations at the application site of treated does exhibited a dose-response pattern of eschar formation, erythema, and edema. The high dose does were the first to show signs of skin irritation and after the treatment stopped, they were the last to show signs of healing. The low and middle dose groups also showed signs of irritation. The edema present during dosing on the does from the 1 % group ceased immediately when the treatment was stopped and the erythema healed completely by gestation day 20. The does at the middle and high dose levels never completely healed from the edema and erythema caused by treatment. The more severe observations at the application site (rippled skin, open sore, black scabs, pus-filled sacs, discoloration and fissuring) appeared on the does from the middle and high does only.

Mortality:

no mortality observed

Description (incidence):

No mortalities occurred throughout the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Although not statistically significant, there was an apparent dose-related trend toward decreased body weight on gestation days 12, 18 and 29. There was also a significant decrease in body weight gain for the does from the high dose group on gestation days 12-18, 6-18 (exposure period) and 6-29. The does from the middle and low dose groups had lower body weight gains than the treated control group, but the values were not statistically significant. The body weight gain during the post exposure period (gestation days 18-29) indicated that the high dose animals gained more weight than the treated controls, but this value was not statistically significant. The untreated control group does had a significantly greater gain in body weight than the treated controls on gestation days 6-12 while the gestation days 12-29 body weight gain was significantly lower than the treated controls. This may indicate that the treatment procedures per se induced stress in pregnant rabbits resulting in a decrease in body weight gain. When treatment ceased, the animals gained more weight than the untreated control which compensated for the initial decrease in weight gain. By sacrifice on gestation day 29, there were no statistically significant differences among groups for body weight. Body weight and corrected body weight at terminal sacrifice were unaffected by treatment.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

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

not specified

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:

effects observed, treatment-related

Description (incidence and severity):

At scheduled sacrifice on gestation day 29, the only statistically significant effect of treatment on the does was an increase in the maternal kidney weight relative to body weight. Although not significant, a dose- response of increased relative liver weight was apparent across the dosed groups.
At terminal sacrifice, there was no treatment-related effect on gravid uterine weight, absolute liver and kidney weights.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination showed effects only in the maternal kidneys where the only treatment related effect was a significant increase of does (20/22) at the high dose and at 5 % (4/22) with vacuolar swelling of the collecting ducts compared with the incidence in the vehicle controls (0/22). The lesion was characterized by a markedly swollen clear cytoplasm within the epithelial cells lining at a particular level of the collecting ducts, with no evidence of concomitant cell degeneration. The lesion was observed almost exclusively in that portion of the ducts present in the outer zone of the inner medulla, i.e. that region where the smaller ducts join to form the larger ducts which pass through the renal papilla.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

not specified

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

>= 2.4 - < 12 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

other: Significant decrease in body weight gain at 24 mg/kg bw/d. Histopathologic changes in the kidneys at 12 and 24 mg/kg bw/d with an increase in incidence of does with vacuolar swelling of the collecting ducts.

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean litter weight and female fetal body weights per litter, taken at sacrifice on gestation day 29, were significantly decreased in the 24 mg/kg bw/day dose group when compared to the treated controls. Female fetal body weight was also decreased at 2.4 mg/kg bw/day but not at 12 mg/kg bw/day. The male fetal body weights per litter did not differ significantly across groups although the male fetal weight at 24 mg/kg bw/day was apparently lower than those in the treated control group.

Reduction in number of live offspring:

not specified

Changes in sex ratio:

not specified

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Mean litter weight, taken at sacrifice on gestation day 29, were significantly decreased in the high dose group when compared to the treated controls.

Changes in postnatal survival:

not specified

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes in the incidence of any external variation. The incidence of litters with one or more fetuses with external variations was significantly elevated at the 2.4 mg/kg bw/day dose group but not at higher doses for ecchymoses on the trunk (but not on the head or extremities).
There was no significant increase in the number of litters with one or more affected fetuses in any exposure group or untreated control group relative to the treated control group for individual and total external, visceral (including cranio-facial) and skeletal malformations, or total malformations (all categories combined) (see Table 2 below).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes in the incidence of any skeletal variations. The incidence of six skeletal findings in the test substance groups differed significantly from that of the treated controls for the following observations the incidence of poorly ossified cervical centrum 1 was elevated at 2.4 mg/kg bw/day (but also in the untreated controls), the incidence of bilobed thoracic centrum 6 was decreased at 2.4 mg/kg bw/day, the incidence of poorly ossified sternebrae 2 and 3 was elevated at 2.4 mg/kg bw/day, the incidence of poorly ossified supraoccipital bone of skull was elevated at 2.4 mg/kg bw/day and the incidence of poorly ossified interparietal bone was elevated at 2.4 mg/kg bw/day. The untreated control group exhibited significant increases in the incidence of two skeletal findings relative to the untreated control group: poorly ossified cervical centrum 1 (mentioned above) and poorly ossified parietal bone of the skull.
There was no significant increase in the number of litters with one or more affected fetuses in any exposure group or untreated control group relative to the treated control group for individual and total external, visceral (including cranio-facial) and skeletal malformations, or total malformations (all categories combined) (see Table 2 below).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes in the incidence of any visceral variations. The incidence of red foci on the thymus was significantly elevated at the 2.4 mg/kg bw/day level. No other statistically significant visceral variations were noted in any dose groups.
There was no significant increase in the number of litters with one or more affected fetuses in any exposure group or untreated control group relative to the treated control group for individual and total external, visceral (including cranio-facial) and skeletal malformations, or total malformations (all categories combined) (see Table 2 below). The middle dose group, however, did exhibit an increase in the number of fetuses and litters with ventricular septal defect (four fetuses in four litters) relative to vehicle controls (one fetus from one liter). This increase was not statistically significant and did not exhibit a dose-response (there was only one fetus at 24 mg/kg bw/day with this malformation). This observation was not considered treatment related.

Other effects:

not specified

Dose descriptor:

NOAEL

Remarks:

fetotoxicity

Effect level:

>= 12 - < 24 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

other: At the 24 mg/kg bw/d dose, there was a significant decrease in mean litter weight and female fetal body weight per litter. At the 24 mg/kg bw/d dose there was a lower (although not significant) male fetal body weight.

Dose descriptor:

NOAEL

Remarks:

teratogenecity

Effect level:

>= 24 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

other: No significant increase in the incidence of individual malformations or of external, visceral, skeletal, or total (external, visceral, plus skeletal) malformations by fetus or by litter in any exposed group.

Dose descriptor:

NOAEL

Remarks:

embryotoxicity

Effect level:

>= 24 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

other: No embryotoxic effects were reported at any dose level studied.

Abnormalities:

effects observed, non-treatment-related

Developmental effects observed:

no

Evaluation of reproductive parameters indicated no treatment related effects on number of ovarian corpora lutea of pregnancy, total implantations, viable or non-viable implantations per litter, percent pre-implantation loss, percent live fetuses per litter, or on sex ratio (% males) (see Table 1).

Table 1. Reproductive Parameters and Fetal Weights in NZW Rabbit Does Exposed to NIAX Catalyst A-99

Parameter	A-99 (volume %, dermally) Gestational Days 6 Through 18
	0.0	Untreated Controls	1.0	5.0	10.0
(Number of does)	(22)	(21)	(21)	(21)	(22)
Corpora lutea per doe	10.0 +/- 1.6^a	9.7 +/- 2.8	9.4 +/- 2.9	8.5 +/- 3.7	9.1 +/- 2.6
Total implants per litter	8.0 +/- 3.0	8.9 +/- 2.4	9.4 +/- 2.0	8.1 +/- 2.5	8.0 +/- 2.6
Percent preimiplantation loss	20.9 +/- 27.0	9.9 +/- 15.6	5.6 +/- 5.3	11.4 +/- 11.7	15.2 +/- 20.9
Viable implantations per litter	7.2 +/- 3.3	7.8 +/- 3.5	8.1 +/- 3.2	7.3 +/- 3.5	7.3 +/- 3.0
Non-viable implants per litter	0.8 +/- 1.5	1.1 +/- 1.4	1.3 +/- 2.3	0.8 +/- 1.7	0.7 +/- 1.5
Early resorptions	0.4 +/- 1.1	0.6 +/- 1.4	0.9 +/- 2.1	0.7 +/- 1.7	0.5 +/- 1.5
Late resorptions	0.0 +/- 0.0	0.0 +/- 0.2	0.0 +/- 0.0	0.0 +/- 0.0	0.0 +/- 0.0
Dead fetuses	0.4 +/- 1.0	0.4 +/- 0.9	0.4 +/- 1.0	0.1 +/- 0.5	0.2 +/- 0.4
Percent live fetuses per litter	88.1 +/- 25.4	80.7 +/- 32.0	84.7 +/- 30.5	83.8 +/- 35.4	90.9 +/- 21.4
Sex Ration (% males)	52.3 +/- 26.4	50.2 +/- 23.1	61.0 +/- 15.9	54.9 +/- 16.2	52.9 +/- 18.2
(Number litters with live fetuses)	(21)	(19)	(19)^b	(18)	(21)
Fetal body weight per litter, g	45.36 +/- 5.82	42.42 +/- 5.08	41.93 +/- 4.00	42.36 +/- 3.19	39.86 +/- 5.13**
Male fetal body weight per litter, g	44.24 +/- 5.23^c	42.50 +/- 3.83^d	42.82 +/- 4.61	43.24 +/- 4.56	40.37 +/- 5.99
Female fetal body weight per litter, g	45.29 +/- 5.90^e	41.95 +/- 5.73	40.92 +/- 4.47*	41.50 +/- 2.83	39.32 +/- 5.32^e**

^a Data presented as mean +/- standard deviation.

^b The sex of one fetus was inadvertently not recorded. Its body weight (34.94 g) was included in the mean litter weight calculations but was not included in fetal body weights by sex.

^c N = 19, two litters had only female fetuses.

^d N = 18, one litter had only female fetuses.

^e N = 20, one litter each at 0.0 and 10.0% had only male fetuses.

* = p < 0.05 versus control (0.0%)

** = p < 0.01 versus control (0.0%)

The type and frequency of fetal malformations observed in the study are provided in Table 2.

Table 2. Malformations Observed in NZW Rabbit Fetuses Exposed to NIAX Catalyst A-99 in Utero^a

A-99 (%, dermally)		Fetuses^b						Litters^c
		0.0	Untreated Controls	1.0	5.0	10.0		0.0	Untreated Controls	1.0	5.0	10.0
NO. EXAMINED EXTERNALLY^d		158	163	170	153	161		21	19	19	18	21
Umbilical hernia	No. (%)	1 (0.6)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.6)		1 (4.8)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.8)
NO. EXAMINED VISCERALLY^e		84	86	88	83	84		21	19	19	18	21
Portion of lower left cerebral hemisphere, missing	No. (%)	0 (0.0)	1 (1.2)	0 (0.0)	0 (0.0)	0 (0.0)		0 (0.0)	1 (5.3)	0 (0.0)	0 (0.0)	0 (0.0)
Dysphagia lusoria (right subclavian artery)	No. (%)	0 (0.0)	0 (0.0)	1 (1.1)	0 (0.0)	0 (0.0)		0 (0.0)	0 (0.0)	1 (5.3)	0 (0.0)	0 (0.0)
Pulmonary artery forms dorsal aorta	No. (%)	1 (1.2)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)		1 (4.8)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Ventricular septal defect	No. (%)	1 (1.2)	0 (0.0)	0 (0.0)	4 (4.8)	1 (1.2)		1 (4.8)	0 (0.0)	0 (0.0)	4 (22.2)	1 (4.8)
Immediate (median) lung lobe, missing	No. (%)	4 (4.8)	0 (0.0)	2 (2.3)	2 (2.4)	0 (0.0)		3 (14.3)	0 (0.0)	1 (5.3)	2 (11.1)	0 (0.0)
Hydronephrosis, unilateral	No. (%)	0 (0.0)	1 (1.2)	0 (0.0)	0 (0.0)	0 (0.0)		0 (0.0)	1 (5.3)	0 (0.0)	0 (0.0)	0 (0.0)
Right testis, missing	No. (%)	1 (1.2)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)		1 (4.8)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Two gallbladders, both smaller than normal	No. (%)	0 (0.0)	0 (0.0)	0 (0.0)	1 (1.2)	0 (0.0)		0 (0.0)	0 (0.0)	0 (0.0)	1 (5.6)	0 (0.0)
Gallbladder and cystc duct, missing	No. (%)	1 (1.2)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)		1 (4.8)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
NO. EXAMINED SKELETALLY^f		74	77	72	70	66		20^g	18^g	18	17	21
Cervical arch #2, only a bone island right side (no anlage)	No. (%)	0 (0.0)	0 (0.0)	1 (1.2)	0 (0.0)	0 (0.0)		0 (0.0)	0 (0.0)	1 (5.3)	0 (0.0)	0 (0.0)
Extra thoracic arch between arches #9 and #10, unilateral	No. (%)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (1.3)		0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.8)
Extra thoracic centrum (partial) between centra #9 and #10 (same fetus as above)	No. (%)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (1.3)		0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.8)
Extra rib between ribs #9 and #10 (with extra arch), (same fetus as above)	No. (%)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (1.3)		0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.8)
NO. EXAMINED SKELETALLY^f		74	77	82	70	77		20^g	18^g	19	18	21
Ribs #5 and #6, fused	No. (%)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (1.3)		0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.8)
Fused ribs (#4 to #5) at distal end, unilateral	No. (%)	0 (0.0)	1 (1.3)	0 (0.0)	0 (0.0)	0 (0.0)		0 (0.0)	1 (5.6)	0 (0.0)	0 (0.0)	0 (0.0)
TOTAL MALFORMATIONS
External Malformations^d	No. (%)	1 (0.6)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.6)		1 (4.8)	0 (0.0)	0 (0.0)	0 (0.0)	1 (4.8)
Visceral Malforamtions^e	No. (%)	7 (8.3)	2 (2.3)	3 (3.4)	7 (8.4)	1 (1.2)		5 (23.8)	2 (10.5)	2 (10.5)	6 (33.3)	1 (4.8)
Skeletal Malformations^f	No. (%)	0 (0.0)	1 (1.3)	1 (1.2)	0 (0.0)	2 (2.6)		0 (0.0)	1 (5.6)	1 (5.3)	0 (0.0)	2 (9.5)
TOTAL MALFORMATIONS	No. (%)	7 (4.4)	3 (1.8)	4 (2.4)	7 (4.6)	4 (2.5)		5 (23.8)	3 (15.8)	3 (15.8)	6 (33.3)	4 (19.0)

^a A single fetus may be represented more than once in listing individual defects.

^b Only live fetuses were examined for malformations.

^c Includes litters with one or more malformed fetuses.

^d All fetuses were examined externally.

^e Approximately 50% of each litter were examined viscerally, and for soft tissue craniofacial malformations.

^f Approximately 50% of each litter were examined for skeletal malformations after staining with Alizarin Red S.

^g One dam had only one live fetus. By convention, it was subjected to a visceral and craniofacial examination.

Table 3. Estimation of Theoretical Dose for 3033 -62 -3

	Estimated Dose (mg/kg bw)
	Assuming Body Weight Satistic Equal to:
Dose	Minimum for Dose Group	Maximum for Dose Group	Average for Dose Group		Overall Minimum	Overall Maximum
(% v/v in water)	GD6-18	GD6-18	GD6-18		GD0	GD0
0	0	0	0		0	0
1	2.4	2.4	2.4		2.9	2.5
5	12	12	12		14	12
10	25	24	24		29	25

Where:

Dose (mg/kg bw) = Dose (% v/v in water) x UC1 x SG x Dw x Vw x UC2 / BW

Unit conversion (UC1): 0.01 [to convert dose in % v/v to mL chem / mL water]

Specific gravity (SG): 0.8696 [=density chem / density water, at 20 deg C as reported in Tyl et al., 1986]

Density water (Dw): 0.998 g/mL = g/cm3, at 20 deg C

Volume water applied to skin (Vw): 1 mL

Unit conversion (UC2): 1000 mg chem/g chem

Body weight (BW): kg [As reported in study report]

Conclusions:

Administration of the test substance by dermal application to timed-pregnant New Zealand white rabbits during organogenesis (gestation days 6-18) resulted in maternal toxicity at 24 and 12 mg/kg bw/day, i.e., transient reduced weight gain during exposure at 24 mg/kg bw/day, renal lesions at 12 and 24 mg/kg bw/day and elevated relative kidney weight at 24 mg/kg bw/day. There was no maternal toxicity at 2.4 mg/kg bw/day except for transient irritation at the application site. Toxicity to the fetus (reduced fetal body weight per litter) was observed unaccompanied by any indication of reduced fetal ossification, only at 24 mg/kg bw/day, a dose which also produced signs of maternal toxicity. There was no evidence for embryotoxicity or teratogenicity at any dose level employed.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Read-accross from related substance

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

>= 2.4 - < 12 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

other: Significant decrease in body weight gain at 24 mg/kg bw/d. Histopathologic changes in the kidneys at 12 mg/kg bw/d and 24 mg/kg bw/d with an increase in incidence of does with vacuolar swelling of the collecting ducts.

Remarks on result:

other: Based on read-across from structural analogue

Key result

Abnormalities:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

fetotoxicity

Effect level:

>= 12 - < 24 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

other: At the 24 mg/kg bw/d, there was a significant decrease in mean litter weight and female fetal body weight per litter. At the 24 mg/kg bw/d dose there was a lower (although not significant) male fetal body weight.

Remarks on result:

other: Based on read-across from structural analogue

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenecity

Effect level:

>= 24 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

other: No significant increase in the incidence of individual malformations or of external, visceral, skeletal, or total (external, visceral, plus skeletal) malformations by fetus or by litter in any exposed group.

Remarks on result:

other: Based on read-across from structural analogue

Key result

Dose descriptor:

NOAEL

Remarks:

embryotoxicity

Effect level:

>= 24 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

other: No embryotoxic effects were reported at any dose level studied.

Remarks on result:

other: Based on read-across from structural analogue

Key result

Abnormalities:

effects observed, non-treatment-related

Key result

Developmental effects observed:

no

Conclusions:

No developmental toxicity study with the test substance is available. Data generated with a structural analogue substance are used for endpoint coverage. A justification for this read-across approach is included in section 13.
Administration of the test substance by dermal application to timed-pregnant New Zealand white rabbits during organogenesis (gestation days 6-18) resulted in maternal toxicity at 24 mg/kg bw/day and 12 mg/kg bw/day, i.e., transient reduced weight gain during exposure at 24 mg/kg bw/day, renal lesions at 12 and 24 mg/kg bw/day and elevated relative kidney weight at 24 mg/kg bw/day. There was no maternal toxicity at 2.4 mg/kg bw/day except for transient irritation at the application site. Toxicity to the fetus (reduced fetal body weight per litter) was observed unaccompanied by any indication of reduced fetal ossification, only at 24 mg/kg bw/day, a dose which also produced signs of maternal toxicity. There was no evidence for embryotoxicity or teratogenicity at any dose level employed.

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:

adverse effect observed

Dose descriptor:

NOAEL

2.4 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Additional information

No developmental toxicity study is performed with the test substance. Data generated with the related substance was used to cover this endpoint.

In a subacute test, Bushy Run Research Center (1985) studied developmental toxicity /teratogenicity (prenatal developmental toxicity study - method equivalent to OECD Guideline 414) via the dermal route of exposure at dose levels of 1, 5 and 10% or 2.4, 12, 24 mg/kg bw/day. Treatment was given once daily at days 6 to 18 of gestation.

There was no evidence for embryotoxicity or teratogenicity at any dose. The maternal toxicity consisted of transient reduced weight gain during exposure at 10% (24 mg/kg bw/day), renal lesions at 5 and 10% (12 -24 mg/kg bw/day) and elevated relative kidney weight at 10% (24 mg/kg bw/day). No adverse maternal toxicity was observed at 1% (2.4 mg/kg bw/day). The principle fetal malformation was reduced fetal body weight per litter without any reduction in fetal ossification except at 10% (24 mg/kg bw/day) a dose at which signs of maternal toxicity were also observed.

Justification for classification or non-classification

The substance is considered not to be classified as reproductive toxicant, according to the criteria laid down in the CLP regulation (EC) No 1272/2008.