Carbamic acid, (2-hydroxypropyl)-, compd. with 1-amino-2-propanol (1:1)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-aminopropan-2-ol hydrochloride

EC Number:

231-948-5

EC Name:

1-aminopropan-2-ol hydrochloride

Cas Number:

7780-04-3

IUPAC Name:

1-aminopropan-2-ol hydrochloride

Constituent 2

Reference substance name:

2-propanol, 1-amino, hydrochloride (1:1)

IUPAC Name:

2-propanol, 1-amino, hydrochloride (1:1)

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany
- Age at study initiation: 11-13 wks
- Weight at study initiation: Males: 267-309 g; Females: 187-218 g
- Housing: individual
- Diet and water: ad libitum (except during the fasting period and measurement of motor activity)
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For preparation of the administration solutions, the test substance was weighed in a graduated measuring flask, toped with tap water and dissolved by shaking. The test substance solutions were prepared at the beginning of the administration period and thereafter at intervals that took into account the stability of the test substance preparation.

VEHICLE
- vehicle: tap water
- pH of dosing solution: 6.0 - 7.5
- Concentration in vehicle: test group 1 - 0; test group 2 - 1.53 g/100 ml; test group 3 - 4.59 g/100 ml; test group 4 - 15.29 g/100 ml
- Amount of vehicle including test substance solution (if gavage): 10 ml
- Purity: calculated value taking into accout the nominal active ingredient content of 65.4g/100ml

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration control analyses were conducted with samples drawn from all doses at the start and at the end of the administration period. Analyses of the test substance dosing solutions in tap water confirmed the targeted concentrations. Generally, the analytical values of the samples corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations

Details on mating procedure:

Each of the male and female animals was mated overnight in a 1 : 1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group.
The animals were paired by placing the female in the cage of the male mating partner from about 4.00 p.m. until 7.00 - 9.00 a.m. of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted "day 0" and the following day "day 1" post coitum.

Duration of treatment / exposure:

Total exposure period: males: 38 days; females: 46 days
Premating exposure period (males and females): 13 days

Frequency of treatment:

once daily

Duration of test:

males: 38 days; females: 46 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw per day
Basis:
actual ingested
(as the hydrochloride salt of isopropanolamine)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

At least 13 days after the beginning of treatment, males and females from the same dose group were mated overnight in a ratio of 1:1 (details of pairing see 3.7.2.). On study day 36, a functional observational battery and motor activity measurement were carried out in the first five male animals per group. Blood from 5 F0 males per group was sampled under Isoflurane anesthesia on study day 38 followed by necropsy of all male animals under CO2 anesthesia. The females were allowed to litter and rear their pups until day 4 after parturition. On study day 43 a functional observational battery and motor activity measurement were carried out in the first five female animals per group. Blood from 5 F0 females per group was sampled under Isoflurane anesthesia on study day 45 followed by necropsy of all female animals and their pups under CO2 anesthesia.

Examinations

Maternal examinations:

BODY WEIGHT / FEED CONSUMPTION / CLINICAL OBSERVATIONS - ADULTS:

A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.

The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g., animal could not litter, umbilical cord not cut) were documented on an individual dam basis. Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable.

Generally, food consumption was determined once a week (in a period of 7 days) for male and female parental animals, with the some minor exceptions. Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning). The body weight change of the animals was calculated from these results.
FUNCTIONAL OBSERVATION BATTERY (FOB) AND MOTOR ACTIVITY TESTING:

A functional observational battery was performed in the first five animals per sex and group at the end of the administration period starting at about 10.00 a.m. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Motor activity was measured on the same day as FOB was performed in the first 5 animal per sex and group. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The animals were put into the cages in a randomized order. The measurements started at about 1.30 p.m.. The number of beam interrupts was counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. Animals received no food and water during the measurements.


CLINICAL PATHOLOGIE:

In the morning, blood was taken from 5 fasted animals per group and sex by puncturing the retroorbital venous plexus under isoflurane anesthesia (Isoflo®, Essex GmbH Munich, Germany). The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. For urinalysis (5/sex/group) the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence.


TERMINATION / GROSS PATHOLOGY / HISTOPATHOLOGY - ADULTS:

The parental animals were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. After the organs were fixed, histotechnical processing and examination by light microscopy were performed.

Ovaries and uterine content:

The ovaries and uterine were examined after termination: Yes
Examinations included:
- Other: organ weight, histopathological examination

Fetal examinations:

CLINICAL OBSERVATIONS / BODY WEIGHT - PUPS:

All pups delivered from the F0 parents were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn pups in each litter. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups. In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on non-workdays.

On the day of birth (day 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups finally confirmed at necropsy. The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. The pups were weighed one day after birth (day 1 post partum) and on day 4 after birth.

TERMINATION / GROSS PATHOLOGY - PUPS:

All surviving pups (after sacrifice on day 4 post partum by means of CO2), all stillborn pups and those pups that died before schedule, were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.
- Organs examined at necropsy (F1 pups): none, gross examination only (external and visceral findings), including those pups that did not survive to sacrifice

Statistics:

Means and standard deviations were calculated. Weight of the anesthetized animals and absolute and relative organ weights were analysed by Kruskal-Wallis and Wilcoxon test.
Further statistical methods used for clinical examination and/or clinical pathology:
DUNNETT, C.W. (1955): A multiple comparison procedure for comparing several treatments with a control. JASA, Vol. 50, 1096 – 1121
DUNNETT, C.W. (1964). New ables for multiple comparisons with a control. Biometrics, Vol. 20, 482 - 491
Siegel S. (1956): Non-parametric statistics for behavioural sciences. McGraw-Hill New York
Nijenhuis, A.; Wilf, H.S. (1978): Combinatorial Algorithms. Academic Press New York, 32-33
Hettmansperger, T.P. (1984); Statistical Inference based on Ranks. John Wiley & Sons New York, 132-142

Indices:

Male mating index, male fertility index, female mating index, female fertility index, gestation index, post implantation loss, live birth index

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: some indication for a mild anemic process in males of the top dose group

Details on maternal toxic effects:
Clinical pathology examinations revealed slightly, but statistically significantly reduced hemoglobin and hematocrit values, which are indicative of a mild anemic process, in high dose males. This is considered as an adverse substance-induced effect. No treatmentrelated effects were noted in hematology investigations of females and serum enzyme examinations of both sexes. Furthermore, reduced urine volumes with subsequently increased specific gravity were excreted by treated animals of either sex. It is likely, that this was caused by a decreased water intake. A reduction in water intake could also well account for the slightly increased urea and albumin levels of the high dose males and/or females. It is concluded that the changes in urinalysis and in blood chemistry examination are not caused by a direct toxic effect of the test compound and are not adverse in nature. This assessment is supported by the fact, that kidney weights as well as gross and histopathological evaluations of this organ did not give any indications for substance-induced alterations.
The test substance did not cause adverse effects regarding terminal body and organ weights, macroscopic evaluation or histopathologic evaluation. The statistically significantly increased liver weights in top dose males and females of the top dose group, which correlated with a minimal to mild diffuse hypertrophy of hepatocytes in several high dose males, mirror adaptive responses to the test substance administration. They are not
assessed as adverse particularly, because liver enzymes in these rats remained unaffected.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Remarks:

for F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

NOAEL

Remarks:

for F0 females

Effect level:

1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

3

Dose descriptor:

NOAEL

Remarks:

for F0 males

Effect level:

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

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No test substance-induced signs of developmental toxicity were noted in the progeny of the F0 parents up to and including 1000 mg/kg body weight/day. The number of delivered F1 pups/litter, their postnatal survival and their body weights remained unaffected by the test substance. Clinical and/or gross necropsy examinations of the F1 pups revealed only findings, which were considered to be spontaneous in nature.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Executive summary:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422) 1-amino-2-propanol was tested as hydrochloride salt to avoid damage to the gastrointestinal tract following gavage administration due to the caustic mode of action. Testing the salt also provides the ability to distinguish between symptoms caused by local effects such as irritation or corrosion and symptoms that are due to systemic toxicity.

1 -Amino-2 -propanol hydrochloride was administered orally via gavage to groups of 12 male and 12 female Wistar rats at doses

of 100, 300, and 1000 mg/kg body weight/day (Schneider, 2005). The study was intended to detect possible effects of the test substance on the integrity and performance of the reproductive system of both sexes and to obtain information about the general toxicological profile after repeated oral administration. The duration of treatment covered a 2 week premating period and a mating period in both sexes, approximately 2 weeks post-mating in males (in total 38 days exposure), and the entire gestation period and 4 days of lactation in females (in total 46 days of exposure).

There were no indications from clinical, gross and histopathological examinations, that the test substance adversely affected fertility or reproductive performance of the F0 parental animals at dose levels as high as 1000 mg/kg body weight/day. Detailed clinical examinations in an open field, detailed observations in a functional observational battery (FOB) and measurements of motor activity did not reveal any indications of test substance-induced effects. Clinical pathology examinations revealed slightly, but statistically significantly reduced hemoglobin and hematocrit values, which are indicative of a mild anemic process, in high dose males only. The test substance did not cause adverse effects regarding terminal body and organ weights, macroscopic evaluation or histopathologic evaluation. The statistically significantly increased liver weights in top dose males and females, which correlated with a minimal to mild diffuse hypertrophy of hepatocytes in several high dose males, mirror adaptive responses to the test substance administration. They are not assessed as adverse particularly, because liver enzymes in these rats remained unaffected.

Under the conditions of this reproduction/developmental toxicity screening test the NOAEL for reproductive performance and fertility is 1000 mg/kg body weight/day for the parental rats. The NOAEL for general, systemic toxicity of the test substance is 300 mg/kg body weight/day for the parental males based on some indications for a mild anemic process. The NOAEL for parental females was found to be 1000 mg/kg body weight/day. The NOAEL for developmental toxicity was 1000 mg/kg body weight/day.