N-isopropylhydroxylamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

A developmental toxicity study was performed to assess the effects of the test item IPHA I-15 Hydroxylamine (IPHA)on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy. The dams (one control and three test item treated groups) were treated daily by oral (gavage) administration, from gestation day GD6 up to and including GD19 (sperm positive day = 0 day of pregnancy, GD0). Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20. The dose levels of 50, 150 and 500 mg/kg bw/day were based on available data including the results of a dose range finding (DRF) study in the pregnant rat.

In the high dose group (500 mg/kgbw/day), there was a clear effect on maternal body weight, weight gain, maternal corrected body weight and on food intake. In the mid dose group (150 mg/kg bw/day) slight effects were seen on body weight gain, maternal corrected body weight and food intake. These effects in the High, and to some extent in the Mid dose group, was considered to reflect a maternal toxicity.There was no evidence of any adverse maternal effects in the low dose group at 50 mg/kgbw/day. There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined up to and including 500 mg/kg bw/day.

There were no effects on the early or late embryonic loss in the test item-treated dams evaluated. No remarkable internal or external observations were recorded for any pregnant animals duringnecropsy. No remarkable abnormalities were observed on the placentas in any examined groups. The mean number of viable foetuses in all test item treated groups as well as their sex distribution were comparable with the control mean. There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined up to and including 500 mg/kg bw/day. The mean foetal body weight per litter was lower and the number of foetuses with retarded body weight was higher, in the high dose group(500 mg/kgbw/day) only, associated with maternal toxicity. There were no direct effects of the test item on external, visceral and/or skeletal development of foetuses in the study. Increased incidence of Ossified sternebra (3 or less)in the mid and high dose group was associated with retarded ossification/development commonly associated with maternal toxicity.

 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May-November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
* Chemical name: Isopropyl hydroxylamine (IPHA)
* Molecular formula: C3H9NO
* CAS number: 5080-22-8
* Batch number: D609G1EGQ9
* Appearance: Colourless liquid
* Purity: 15% IPHA aqueous solution
- Expiration date of the lot/batch: 14 January 2017
- Purity test date:14 January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, under inert gas (15-25 ⁰C, below 70 RH%)
- Safety Precautions: Routine safety precautions (lab coat, gloves, safety glasses and face mask) for unknown materials will be applied to assure personnel health and safety. Dangerous to the environment
- Stability under test conditions: test Item formulations in distilled water at concentrations 10 and 100 mg/mL are stable in room temperature for 11 days.
- Solubility and stability of the test substance in the solvent/vehicle: the test Item is supplied as a 15% solution in water and the applied vehicle is distilled water.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item will be formulated in distilled water at the appropriate concentrations according to the dose levels selected. During the dose formulation the pH of each concentration will be adjusted to below pH 10, the added volume of 10% HCl solution used for adjustment and the final pH value of the formulations will be documented in the raw data and reported.
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species and strain: Hannover Wistar rats (CRLHan)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Justification of strain: The rat is regarded as a suitable rodent species for reproduction studies and the test guideline states it is the preferred rodent species. The Hannover Wistar rat was selected due to experience with this strain in teratology studies at CiToxLAB Hungary Ltd.
- Age at study initiation: young adult female rats, nulliparous and non-pregnant, at least approximately 11 weeks old
- Weight at study initiation: not to exceed ± 20% of the mean weight at onset of treatment
- Fasting period before study:
- Housing/Enrichment: Rodents will be housed individually. Nest building material (ARBOCEL natural crinklets produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, Rosenberg, D-73494 Germany), or similar will be also added to the cages.
- Diet (e.g. ad libitum): The animals will be provided with ssniff® SM R/M-Z+H “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (ssniff Spezialdiäten GmbH, D-59494 Soest, Germany), ad libitum. The diet is routinely analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Water (e.g. ad libitum): tap water as for human consumption, ad libitum. The quality control analysis of the water is performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36., Hungary).
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
The temperature and relative humidity values are measured twice daily during the acclimatisation period and during the experiment.
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

The oestrus cycle of female animals wwas examined shortly before start of pairing. After acclimatisation the females, according to their oestrus cycle, were paired with males for approximately 2 hours (1 male: 1-3 females) in the morning, until at least 24 sperm-positive females/group were obtained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (GD0). The sperm-positive, assumed pregnant females were allocated to each experimental group (on each mating day) in such a way that the group averages of the body weight were as similar as possible.

Frequency of treatment:

The control or test item dose formulations were administered to mated, sperm positive (assumed pregnant) female rats daily by oral gavage on a 7 days/week basis, approximately at similar times, from Gestation Day (GD) 6 to GD19.

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 mated female animals/group, 4 groups (one control and 3 test item-treated groups)

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

In the Dose Range Finding (DRF) study doses of 125, 250 and 500 mg/kg bw/day were administered by oral gavage to pregnant rats from GD6 to GD19 [4]. The number of evaluated animals in the DRF study was 6 (125, 250 and 500 mg/kg bw/day dose groups) or 5 (control group). Dose of 500 mg/kg bw/day produced maternal toxicity (evident in decreased body weight, decreased body weight gain and decreased food consumption). No signs of embryotoxicity or foetotoxicity were observed in any test item treated dose groups (up to and including 500 mg/kg bw/day). Based on the above results, doses of 50, 150 and 500 mg/kg bw/day were selected for the main study

Maternal examinations:

Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). Cage-side clinical observations were made at twice daily (at the beginning and end of each working day). Detailed clinical observations were made on all animals at the onset of treatment (GD6) then weekly. The animals were monitored for any changes including pertinent behavioural changes and signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. On GD13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).

Ovaries and uterine content:

Before expected delivery, on GD20, Caesarean section was performed on each treated dam. The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes. The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses. The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted, the number and percentage of pre- and postimplantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.

Fetal examinations:

Each live foetus was weighed individually (accuracy ±0.01 g) and subjected to external examination, plus an additional examination of the great arteries. The gender of foetuses was determined according to the anogenital distance. Thereafter the foetuses were individually identified; approximately half of each litter was subjected to visceral examination, and the other half was processed for skeletal examination. For the foetuses subjected to visceral examination, the abdominal and thoracic region was opened and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sanomya mixture then after fixation the body was micro-dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. For the foetuses subjected to skeletal examination, the abdominal region was opened, and the viscera and skin of foetuses were removed and the cadaver was fixed in alcian-blue - acetic acid - ethanol mixture. After fixation in isopropanol the skeletons were stained by KOH-Alizarin red-S method and examined by means of a dissecting microscope. All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded; additionally photographic records were made.

Statistics:

Data were collected using the software PROVANTIS v.9 (maternal data and necropsy). The statistical evaluation of data was performed with the program package SAS 9.2 in case of Provantis 9, or SPSS PC+4.0 (SPSS Hungary Kft, Budapest) in the case of data tabulated in Excel, by an appropriate statistical method.

Data were collected to provide information on parameters including:

Maternal Data:
- Number of animals at test start, no. of animals surviving, no. of pregnant animals, no. of animals with total intrauterine mortality
- Clinical signs (by gestation day)
- Mortality (by gestation day)
- Body weight and body weight gain: mean ± S.D.
- Corrected body weight on GD20 (body weight-gravid uterine weight) and corrected body weight gain: mean ± S.D.
- Net body weight change (Body weight on GD20 minus body weight on GD6 minus gravid uterine weight): mean ± S.D.
- Food consumption: mean ± S.D.
- Gross pathology findings
- Gravid uterine weight

Caesarean Section and Necropsy Data:
- Number of corpora lutea: mean ± S.D.
- Number of implantations: mean ± S.D.
- Number and percentage of live foetuses: mean ± S.D.
- Number and percentage of intrauterine mortality: mean ± S.D. Classified according to time of death: preimplantation loss, postimplantation mortality, Early and late embryonic, as well as foetal death

Indices:

- Preimplantation loss: %, group mean
(Number of corpora lutea-Number of implantations x100) / Number of corpora lutea

- Postimplantation loss: %, group mean
(Number of implantations-Number of live foetuses) x100 / Number of implantations

Foetal Data:

- Sex distribution: %, group mean
(Number of male (female) foetuses) x100 / Number of foetuses

- Foetal body weight (accuracy 0.01 g): mean * S.D.

- External abnormalities/litter: %, group mean(
Number of foetuses with abnormality) x100 / Number of foetuses

- Visceral abnormalities/litter: %, group mean
(Number of foetuses with abnormality) x100 / Number of foetuses

- Skeletal abnormalities/litter: %, group mean
(Number of foetuses with abnormality) x100 / Number of foetuses

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related adverse effects or systemic clinical signs were noted in the treated animals.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the high-dose group (500 mg/kg bw/day), there was a clear effect on maternal body weight, weight gain, maternal corrected body weight and on food intake. In the mid-dose group (150 mg/kg bw/day) slight effects were seen on body weight gain, maternal corrected body weight and food intake. These effects in the high, and to some extent in the Mid dose group, was considered to reflect a maternal toxicity. There was no evidence of any adverse maternal effects in the low dose group at 50 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean daily food consumption of dams in the mid- and high-dose groups (150 and 500 mg/kg bw/day) from the onset of the treatment was below of the control group by approximately 6.1-40.3%, attaining statistical significance between GD 6-8, 8-10, 12-14, 16-18 and, in the case of the high dose group, between GD 18-20. The overall food consumption data in these groups was also below the control value by approximately 10.2% and 24.6 between GD 6-20, and 8.0 and 19.3% between 0-20, respectively. The difference observed was statistically significant in both cases (at p < 0.01 level). Although there was a minor, non-significant difference between these two and the control groups before the start of the treatment, this effect on the food consumption of the mid- and high dose group (150 and 500 mg/kg bw/day, respectively) was considered to be treatment related.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

No observations were recorded for any animals in the study.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The weight of foetuses per litter in the low- and mid-dose groups (50 and 150 mg/kg bw/day, respectively) did not differ significantly from the control mean value. In the high (500 mg/kg bw/day) dose group, foetus weight per litter was significantly (p < 0.01) lower compared to the control group. The weight difference in this group is considered to be test item related but is compatible with an indirect effect of maternal toxicity.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

The weight of foetuses per litter in the low- and mid dose groups (50 and 150 mg/kg bw/day, respectively) did not differ significantly from the control mean value. In the high (500 mg/kg bw/day) dose group, foetus weight per litter was significantly (p < 0.01) lower compared to the control group. The weight difference in this group is considered to be test item related but is compatible with an indirect effect of maternal toxicity. The total number of retarded foetuses (runts) as well as the number of affected litters was higher in the high dose group compared to the control group, indicating retarded body development, probably secondary to maternal toxicity.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were no malformations in the mid- or high groups, a low incidence (2/124 foetuses) in the low-dose was ascribed to incidental background findings, unrelated to treatment.

Visceral malformations:

no effects observed

Description (incidence and severity):

All of the visceral findings are consistent in general nature and incidence with the study concurrent control data or the existing historical control data, therefore considered as incidental findings. Ossified sternebra (3 or less) with 4/22 and 10/21 litters affected in the mid- and high-dose respectively; the high dose group was clearly affected by treatment, the mid-dose level individual incidence (8/116 foetuses) was slightly above the normal range and was statistically significant (p<0.05). This finding is considered to be related to retarded ossification, a common foetal finding in the presence of maternal toxicity.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined up to and including 500 mg/kg bw/day. The mean foetal body weight per litter was lower and the number of foetuses with retarded body weight were higher, in the high-dose group (500 mg/kg bw/day) only, associated with maternal toxicity. There were no direct effects of the test item on external, visceral and/or skeletal development of foetuses in the study. Increased incidence of ossified sternebra (3 or less) in the mid- and high dose group was associated with retarded ossification/development commonly associated with maternal toxicity.

Key result

Dose descriptor:

NOAEL

Effect level:

> 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effects observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

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

Justification for classification or non-classification

According to the criteria of Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, IPHA is not classificable for developmental toxicity.