Reaction mass of 2-aminoethanol and 6-[(p-tosyl)amino]hexanoic acid, compound with 2-aminoethanol (1:1)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

toxicity to reproduction

Remarks:

other: screening

Type of information:

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

Adequacy of study:

key study

Study period:

From 23 May 2012 to 30 September 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 422. The study was conducted on 6-[(p-Tosyl)amino]hexanoic acid, which is the carboxylic acid component of the registered substance.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River, Germany, D-97633 Sulzfeld- Age at purchase: 11 weeks - weight rage at time of grouping: males, 175-200 g- Fasting period before study: no- Housing: 2 per cage, - Cages: TECHNIPLAST filter top cages type 2145 F with an G-Temp (PSU) durable filter cover, 480x265x210 mm³, floor area 940 cm²,- Source: Techniplast Company, Italy- Diet: ad libitum, M3, BONAGRO Ltd., reg. CZ 10174, Czech Republic- Water: ad libitum, tap water- Acclimation period: 9 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 22 +/- 2- Humidity (%): 40 - 70- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:VEHICLE: suspension in water- Amount of vehicle: 10 ml/kg at similar times each day

Details on mating procedure:

- Impregnation procedure: cohoused- If cohoused: - M/F ratio per cage: 1/1 (probably) (male animals: see Endpoint study record in section 7.5.1 "Repeated dose Toxicity") - Length of cohabitation: until copulation/ up to 14 days- Verification of same strain and source of both sexes: yes - Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy- M/F ratio per cage: 1/1- Length of cohabitation: Each morning the females were examined for the presence of sperm in vaginal lavages- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy- After successful mating each pregnant female was caged individually.- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

males: 54 administrations, (2 weeks before mating, 2 weeks during mating and 26 days after mating period)females: 14 days pre-mating period and until mating, through the pregnancy period (assuming 20 days pregnancy) until lactation day 3 after delivery (4 days lactation period)

Frequency of treatment:

daily by stomach tube

Details on study schedule:

- Age at mating of the mated animals in the study: 14 weeks

Remarks:

Doses / Concentrations:actual ingested dosesBasis:actual ingested0, 100, 400, 1600 mg/kg bw./day

No. of animals per sex per dose:

12 males and 12 females per group including control group. 5 males and 5 females per satellite group.

Control animals:

yes

Details on study design:

- Control groups: drinking water- Dose selection rationale: Results of an acute toxicity study with oral administration to male and female rats- Result: no effects up to and including 2000 mg/kg bw.- Rationale for animal assignment: randomly grouped- Section schedule rationale: all animals were sacrificed

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes - Time schedule: dailyBODY WEIGHT: Yes - Time schedule for examinations: on administration days 1, 8, 15, 22, 29, 36, 42, 50 and on day of autopsy, pups within 24 hours of parturition and on day 4 post-partumFOOD CONSUMPTION : 2 weeks before mating, after 2 week mating period and weekly until the end of the studyHAEMATOLOGY: Yes - Time schedule for collection of blood: day 14 (before mating) and prior to autopsy from the satellite groups and from the control and high dose group.CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood:day 14 (before mating) and prior to autopsy from the satellite groups and from the control and high dose group.URINALYSIS: No

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight, histopathology of testis and epidymidis

Litter observations:

PARAMETERS EXAMINEDnumbers of pups born, delivery index, number of pups alive, birth index, live birth index, pup weight on day 0 of lactation, sex ratio, number of pups alive on day 4 of lactation, pup weight on day 4 of lactation, general status of pups, observation of external deformities, corpora lutea, implantations, visible resorptions, early resorptionsGROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities

Postmortem examinations (parental animals):

POST-MORTEM EXAMINATIONS: Yes SACRIFICE- Male animals after 54 days: All surviving animals- Female animals: Sacrifice on lactation day 4 (starting with day 0),ORGAN WEIGHTS- Organ weights: brain, heart, thymus, spleen, liver, testis, epididymis, kidney, adrenal glandHISTOPATHOLOGY: Yes: high dose and control animals- Organ: medulla oblongata, brain, heart, pancreas + lymphnode, spleen, liver, lung, small intestine, stomach, kidneys, adrenal gland, testes, prostrate, urinary bladder, bone + bone marrow, thymus, trachea, white + brown fat, muscle, pituitary gland

Postmortem examinations (offspring):

Gross pathology: observation of external deformities

Statistics:

Statistical evaluation was operated using the software SPPS version 16.0. Group data were represented by mean, standard deviation and median. Statistical analysis in case of data measured once during the study (organ weight, haematology, clinical chemistry) : Mann-Whitney U test for pairwise comparison between control and individual experimental groups on significance level alpha = 0.05.Statistical analysis in case of repeated data measurement (body weight, food intake): Repeated measures ANOVA (procedure General Linear Model (GLM) for Repeated Measures in SPSS statistical software).

Reproductive indices:

Number of mated pairs, number of copulated pairs, copulation index, number of pregnant animals, fertility index, pairing days until copulation, frequency of vaginal estrus, number of pregnant females, number of pregnant females with pups alive, gestation index, gestation length in days, number of corpora lutea, number of implantation sites, implantation index, number of pups born, delivery index

Offspring viability indices:

Number of pups alive, birth index, live birth index, pup weight on day 0 of lactation, sex ratio, number of pups alive on day 4 of lactation, viability index, pup weight on day 4 of lactation, general status of pups, observation of external deformities

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS:- 100, 400 and 1600 mg/kg bw/day: no differences to the control animals observableMORTALITY: noBODY WEIGHT GAIN: 1600 mg/kg bw: reduced FOOD CONSUMPTION: no statistical differencesHAEMATOLOGY/ CLINICAL CHEMISTRY:Haematology and clinical chemistry reveals some statistically significant differences, but these were not related to dosage or not confirmed by the findings in other groups, for example by the results of the satellite groups, or the effects are of biological low relevance i.e..URINALYSIS: not examinedNEUROBEHAVIOUR: not examinedORGAN WEIGHTS: no statistical differencesGROSS PATHOLOGY: no dosage related effectsHISTOPATHOLOGY: NON-NEOPLASTIC: no statistical differencesHISTOPATHOLOGY: NEOPLASTIC: no statistical differencesHISTORICAL CONTROL DATA: not given

Dose descriptor:

NOEL

Remarks:

fertility

Effect level:

>= 1 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Considered to be the NOAEL for systemic and reproduction-developmental toxicity.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: See 'Remark'

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

CLINICAL SIGNS (OFFSPRING): no abnormalities in the general statusBODY WEIGHT (OFFSPRING): no significant differences between the control group and the various dosage groups were observed in males or females in body weights on day 0 On lactation day 4 a higher number of dead pups was counted in the high-dose level group. Moreover, there was a statistically significant but very low difference in the body weight between the control group and the 400 and the 1600 mg/kg bw dose groups. However, this difference is considered to be of low biological relevance.GROSS PATHOLOGY (OFFSPRING): no external deformities were observed in the surviving pups on the day of birth. In autopsies of pups on lactation day 4, no abnormalities were observed in the control, nor were any abnormalities in the autopsy of the dead pup.

Key result

Dose descriptor:

NOEL

Remarks:

fertility

Generation:

F1

Effect level:

> 1 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: See 'Remark'

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

Daily dosages of 0, 100, 400, and 1600 mg ASC plus/kg bw. were administered by stomach tube to groups of 12 male and female rats. Treatment of males: 54 administrations, (2 weeks before mating, 2 weeks during mating and 26 days after mating period), treatment of females: 14 days pre-mating period and until mating, through the pregnancy period (assuming 20 days pregnancy) until lactation day 3 after delivery (4 days lactation period) for 54 days. Additional satellite groups of 5 male and 5 female were treated with 0 and 1600 mg ASC plus/kg bw. and used for observation of reversibility, persistence or delayed occurrence of systemic toxic effects for at least 14 days post treatment.The test-article was formulated in drinking water and administered in 10 ml/kg bw..At dosages of 100 and 400 mg/kg bw. the animals showed no differences to the control animals (NOAEL).1600 mg ASC plus/kg bw. may have induced an influence on the body weight gain of the males and the survival of pups until day 4 after birth.NOAEL: 400 mg/kg bw. However NOAEL (Fertility) = 1600 mg/kg bw.

Executive summary:

The toxicity of the test substance to reproduction was determined in accordance with the OECD Guideline for Testing of Chemicals 422. Dose of 0, 100, 400, and 1600 mg/kg bw/day of the test substance were administered by stomach tube to groups of 12 male rats for 54 days. The test-article was formulated in drinking water and administered in 10 ml/kg bw. Histopathology and gross necropsy did not reveal any test article related changes. The organ weights showed some statistically significant differences, but these were not related to dosage or not affirmed by the findings in other groups, for example by the results of the satellite groups.

The same is true for the statistical significant differences of the haematological parameters and the result of the clinical chemistry, especially decreased activities of the liver enzymes in the blood. These are merely statistical effects without biological relevance. Two effects with a possible relation to the oral administration of the test substance were observed:

- At birth no differences were observed between the groups, but on day 4 after birth a higher number of dead pups was counted in the high-dose level group.

- With male rats a dose dependent decrease in body weight is observed. This effect is in the highest dose group statistically significant and confirmed by the satellite group but not confirmed by the body weight development of the females and not confirmed by the food consumption of males and females.

Although the influence of an infection by a parasite could be a possible explanation of both effects, a non-specific influence of the test substance cannot be excluded. The daily administration of a suspension of the test substance in 10 ml/ kg bw may have led to a reduced appetite of the animals. This could be especially true for the highest dosage group, as here the suspension was relatively dense. It is also possible that there was an osmotic effect of non-resorbed test substance in the intestine. At dosages of 100 and 400 mg/kg bw the animals showed no differences to the control animals. A dose of 1600 mg/kg bw/day of the test substance may have induced an influence on the body weight gain of the males and the survival of pups until day 4 after birth. With regard to effects on fertility of the present study 1600 mg/kg bw/day of the test substance can be defined as the NOAEL. The NOAEL for systemic toxicity in the P and F1 generation of test animals was found to be 400 mg/kg bw/day.

The test substance was the carboxylic acid component of the Reaction mass of 2-aminoethanol and 6-[(p-tosyl)amino]hexanoic acid, compound with 2-aminoethanol (1:1).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The potential of the registered substance to be toxic to the reproduction is considered to be driven by the potential of its constituents, namely 2 -aminoethanol, 6-[(p-tosyl)amino]hexanoic acid, and water.

In accordance with Annex VIII of REACH, a screening is recommended in order to asses this endpoint for the tonnage band of the substance, unless a higher-tier study is available.

The toxicity of the 6-[(p-tosyl)amino]hexanoic acid to reproduction was determined in accordance with the OECD Guideline for Testing of Chemicals 422. Dose of 0, 100, 400, and 1600 mg/kg bw/day of the test substance were administered by stomach tube to groups of 12 male rats for 54 days. The test-article was formulated in drinking water and administered in 10 ml/kg bw. Histopathology and gross necropsy did not reveal any test article related changes. The organ weights showed some statistically significant differences, but these were not related to dosage or not affirmed by the findings in other groups, for example by the results of the satellite groups.

The same is true for the statistical significant differences of the haematological parameters and the result of the clinical chemistry, especially decreased activities of the liver enzymes in the blood. These are merely statistical effects without biological relevance. Two effects with a possible relation to the oral administration of the test substance were observed:

- At birth no differences were observed between the groups, but on day 4 after birth a higher number of dead pups was counted in the high-dose level group.

- With male rats a dose dependent decrease in body weight is observed. This effect is in the highest dose group statistically significant and confirmed by the satellite group but not confirmed by the body weight development of the females and not confirmed by the food consumption of males and females.

Although the influence of an infection by a parasite could be a possible explanation of both effects, a non-specific influence of the test substance cannot be excluded. The daily administration of a suspension of the test substance in 10 ml/ kg bw may have led to a reduced appetite of the animals. This could be especially true for the highest dosage group, as here the suspension was relatively dense. It is also possible that there was an osmotic effect of non-resorbed test substance in the intestine. At dosages of 100 and 400 mg/kg bw the animals showed no differences to the control animals. A dose of 1600 mg/kg bw/day of the test substance may have induced an influence on the body weight gain of the males and the survival of pups until day 4 after birth. With regard to effects on fertility of the present study 1600 mg/kg bw/day of the test substance can be defined as the NOAEL. The NOAEL for systemic toxicity in the P and F1 generation of test animals was found to be 400 mg/kg bw/day.

The prenatal developmental toxicity of 2-aminoethanol was determined according to a method similar to the OECD Guideline for Testing of chemicals 414 with doses of 0, 40, 120 and 450 mg/kg bw/day. The developmental toxicity of the substance dispersed in water was studied in rats following a daily exposure through day 6 to 15 of gestation during the organogenesis. A preliminary study was performed in order to determine a highest dose that would induce some maternal toxicity but not death or severe suffering.

25 animals per dose were sacrificed at Day 20 of gestation - one day before the parturition - while 15 animals per dose were allowed to deliver. Remaining parents and pups were sacrificed at Day 21 post-partum at the end of the lactation.

Body weights were recorded on days 0, 1, 3, 6, 8, 10, 13, 15, 17, and 20 of gestation, and days 4, 7, 14, and 21 post-partum. All animals were observed daily for appearance and behaviour. Food consumption was determined every 2-3 days.

Sacrificed animals were all subject to post-mortem examination. Gravid animals were examined from uterus weight, and number of corpora lutea, implantations, and resorptions. Fetuses underwent external examination, along with soft tissue examination. Half were stained with alizarin red-S for skeletal examination.

Animals delivered by the surviving females were examined for liveborn and stillborn, sexed, weighed on Day 1, 4, 7, 14, and 21 post-partum. Litters were examined daily for mortality and clinical symptoms. All animals were examined for external and internal examination.

In accordance with the preliminary testing, the highest dose of 450 mg/kg bw/day induced some maternal toxicity with a significant reduction of the food consumption, which led to a significant reduction in body weight and body weight gain. No other significant effects were reported for any dose. It was concluded that the NOAEL (developmental toxicity) was 450 mg/kg bw/day, as the highest dose tested had no significant effects.

Read-across between the carboxylic acid (6-[(p-Tosyl)amino]hexanoic acid) and the registered substance is considered justified as the registered substance is manufactured directly from 6-[(p-Tosyl)amino]hexanoic acid by simple neutralisation with 2-aminoethanol in water. In water as they are in the reaction mass, the acid and amine components of the Reaction mass of 2-aminoethanol and 6-[(p-tosyl)amino]hexanoic acid, compound with 2-aminoethanol (1:1) are completely dissociated and behave essentially as independent substances.

Toxicity of 2-aminoethanol is local and due to its high pH value. In the reaction mass, it acts as a pH-control for the 6-[(p-Tosyl)amino]hexanoic acid, so no local toxicity is expected. No long-term systemic toxicity is expected as the substance will rapidly metabolise with NH3 and acetyl-CoA as its final degradation products. Therefore, it is considered that the acid component of the reaction mass will have a more significant impact on the outcome of any (eco)toxicological or environmental tests. The pKa of the carboxylic acid group in the reaction mass. As a result, 6-[(p-Tosyl)amino]hexanoic acid will respond to changes of pH in the same way whether it is in the salt form or as the parent carboxylic acid and hence its bioavailability will be the same.

As 6-[(p-tosyl)amino]hexanoic acid, the registered substance is not considered to be toxic to reproduction, the registered substance does not need to be classified as such according to EU CLP criteria.

Short description of key information:
The toxicity to reproduction of the carboxylic acid component of the registered substance was determined in accordance with the OECD Guideline for Testing of Chemicals 422. It was concluded that the NOAEL (fertility) was 1600 mg/kg bw/day. NOAEL (systemic) for P animals and F1 animals was 400 mg/kg bw/day.

In accordance with Annex VIII of REACH, a screening for reproductive/developmental toxicity is required unless a higher tier reproductive toxicity study is available. The toxicity to the reproduction of the ethanolamine component of the registered substance was determined according to a method similar to OECD Guideline for Testing of Chemicals 414. It was concluded that the NOAEL (developmental toxicity) was 450 mg/kg bw/day, which was the highest dose tested as selected from a preliminary study to induce some maternal toxicity. NOAEL (maternal toxicity) for P animals and F1 animals was 120 mg/kg bw/day.

As the toxicity to the reproduction of the registered substance is considered to be driven by the toxicity to the reproduction on ifs constituents - namely 2-aminoethanol, 6-[(p-tosyl)amino]hexanoic acid, and water - the registered substance did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Justification for selection of Effect on fertility via oral route:
Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 422. The study was conducted on the carboxylic acid component of the registered substance.

Justification for selection of Effect on fertility via inhalation route:
In accordance with REACH, Annex XI, no testing is required as no significant inhalation exposure is expected in all scenarios of the manufacture and all identified uses. The substance is only associated with industrial uses and is handled by professional users with protective equipment in controlled conditions. Moreover, the substance will only be used in diluted form.

Justification for selection of Effect on fertility via dermal route:
In accordance with REACH, Annex XI, no testing is required as no significant dermal exposure is expected in all scenarios of the manufacture and all identified uses. The substance is only associated with industrial uses and is handled by professional users with protective equipment in controlled conditions. Moreover, the substance will only be used in diluted form.

Justification for classification or non-classification

Read-across between the carboxylic acid (6-[(p-Tosyl)amino]hexanoic acid) and the registered substance is considered justified as the registered substance is manufactured directly from 6-[(p-Tosyl)amino]hexanoic acid by simple neutralisation with 2-aminoethanol in water. In water as they are in the reaction mass, the acid and amine components of the Reaction mass of 2-aminoethanol and 6-[(p-tosyl)amino]hexanoic acid, compound with 2-aminoethanol (1:1) are completely dissociated and behave essentially as independent substances.

Toxicity of 2-aminoethanol is local and due to its high pH value. In the reaction mass, it acts as a pH-control for the 6-[(p-Tosyl)amino]hexanoic acid, so no local toxicity is expected. No long-term systemic toxicity is expected as the substance will rapidly metabolise with NH3 and acetyl-CoA as its final degradation products. Therefore, it is considered that the acid component of the reaction mass will have a more significant impact on the outcome of any (eco)toxicological or environmental tests. The pKa of the carboxylic acid group in the reaction mass. As a result, 6-[(p-Tosyl)amino]hexanoic acid will respond to changes of pH in the same way whether it is in the salt form or as the parent carboxylic acid and hence its bioavailability will be the same.

As 6-[(p-tosyl)amino]hexanoic acid, the registered substance is not considered to be toxic to reproduction, the registered substance does not need to be classified as such according to EU CLP criteria