12-aminododecanoic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 October 2006 - 04 April 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

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:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Strain: Sprague-Dawley
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: approximately 8 weeks.
- Weight at study initiation: males: 257 - 318 g; females: 198 - 255 g.
- Housing: Initially, all animals were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. During the mating phase for the non-recovery dose groups, animals were transferred to similar cages on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes (Datesand Ltd. Cheshire, UK). Recovery animals were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper.
- Diet (e.g. ad libitum): ad libitum. A pelleted diet (Rodent PMI 5002 (Certified) diet, BCM IPS Limited, London, UK) was used throughout the study.
- Water (e.g. ad libitum): ad libitum. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 14 days.

The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
Environmental enrichment was provided in the form of wooden chew blocks (B & K Universal Ltd, Hull, UK) and cardboard fun tunnels (Datesand Ltd, Cheshire, UK) except for mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 15% relative
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

The stability and homogeneity of the test material formulations were determined analytically and show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately at 4°C in the dark
The vehicle was chosen due to the complex nature of the test material and its limited solubility in organic and aqueous media.

Treatment Group Dose Level Treatment Volume Concentration
(mg/kg/day) (mL/kg) (mg/mL)
Control 0 4 0
Low 50 4 12.5
Intermediate 250 4 62.5
High 1000 4 250
Recovery Control 0 4 0
Recovery High 1000 4 250

The test material was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg/day of Arachis oil BP.

The volume of test and control material administered to each animal was based on the most recent scheduled bodyweight and was adjusted at regular intervals.

Details on mating procedure:

Initially, all animals were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. During the mating phase for the non-recovery dose groups, animals were transferred to similar cages on a one male: one female basis. Following evidence of mating using vaginal smearing, the males were returned to their original cages and females were transferred to individual cages.
Non-recovery animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical Analysis of Test Material Formulations, Methods and Results
METHOD OF ANALYSIS
Due to the complex nature of the test material and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed. The concentration of 12-Aminododecanoic acid in the test material formulations was determined using a gravimetric technique.

-Samples
The test material formulations were weighed into tared glass sintered crucibles and then rinsed with acetone to leave a test material residue. The samples were then dried in an oven at approximately 105 °C before being allowed to cool over silica gel in a desiccator and re-weighed.
-Homogeneity Determinations
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.
-Stability Determinations
The test material formulations were sampled and analysed initially and then after storage at approximately +4 °C in the dark for fourteen days.
The analytical procedure is considered to be non stability indicating, however the residue was subjected to an FTIR scan and the results suggest the test material is stable in the formulation.
-Verification of Test Material Formulation Concentrations
The test material formulations were sampled and analysed within two days of preparation.

Homogeneity of Test Material Formulations:

Nominal Concentration Sampling Location Concentration Found (mg/mL)
(mg/mL) 1 2 3 Mean
Top 13.2 12.7 13.4 13.1
12.5 Middle 11.5 13.1 13.2 12.6
Bottom 13.5 13.1 13.4 13.3
Top 250 245 246 247
250 Middle 246 242 236 241
Bottom 248 235 224 236


Stability of Test Material Formulations:

Nominal Concentration Concentration Found Initially Concentration Found After Storage for Fourteen Days
(mg/mL) (mg/mL) (mg/mL) (expressed as % of initial)
12.5 13.0 11.5 88
250 241 248 103


Verification of Concentration of Weekly Test Material Formulation:

Week No. Nominal Concentration Concentration Found
(mg/mL) (mg/mL) (expressed as % of nominal)
0 ND -
12.5 12.1 97
1 62.5 58.8 94
250 243 97

0 ND -
12.5 12.7 101
2 62.5 61.1 98
250 246 98

0 ND -
12.5 12.7 99
3 62.5 62.0 99
250 245 98

0 ND -
12.5 12.9 103
4 62.5 65.3 104
250 248 99

0 ND -
12.5 12.5 100
5 62.5 61.0 98
250 246 98

0 ND -
12.5 13.3 107
6 62.5 64.0 102
250 246 98

0 ND -
12.5 10.8 87
7 62.5 61.2 98
250 241 96

ND = none detected
- = not applicable

METHOD VALIDATION
Specificity
A blank Arachis Oil BP (control) was analysed. The analysis of the blank Arachis Oil BP (control) produced no signal that interfered with the signal due to the test material.

Accuracy
Samples of Arachis Oil BP were accurately fortified with known amounts of test material, and analysed:
Fortification Concentration Found Recovered Mean Recovery
(mg/g) (mg/g) (%) (%)
12.6 12.9 103 103
12.9 13.6 104
244 234 96 97
244 239 98
The analytical method has been considered to be sufficiently accurate for the purpose of this study. The test sample results have not been corrected for recovery.

Conclusion
The analytical method has been satisfactorily validated in terms of specificity and accuracy for the purposes of the study.

Duration of treatment / exposure:

Up to 54 days (treatment groups) and 42 days (recovery groups).

Frequency of treatment:

Daily

Details on study schedule:

On day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of 14 days.

Remarks:

Doses / Concentrations:
0, 50, 250 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex per dose in the treatment groups.
5 animals per sex per dose in the recovery groups.

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were chosen based on the results of a preliminary range-finder.

The test material was administered by gavage to three groups each of ten male and ten female rats for up to fifty-four consecutive days, at dose levels of 50, 250 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for forty-two consecutive days and then maintained without treatment for a further fourteen days.
Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating and at termination on five selected non-recovery males and females from each dose group. In addition, haematology and blood chemistry were evaluated after the fourteen day treatment free period on all recovery animals. Urinalysis was evaluated on five selected non-recovery males at the end of the treatment period and for all recovery group males at the end of the treatment free period.

Parental animals: Observations and examinations:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before and after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). During the treatment-free period, recovery animals were observed twice daily (once daily at weekends). All observations were recorded.

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females per non-recovery dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose-built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin colour
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behaviour Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation

Functional Performance Tests
-Motor Activity: Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period).

-Forelimb/Hindlimb Grips Strength: An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

- Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli:
Grasp response Touch escape
Vocalisation Pupil reflex
Toe pinch Startle reflex
Tail pinch Blink reflex
Finger approach

-Bodyweight
Individual bodyweights were recorded on Day 0 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination.

-Food Consumption
During the maturation period, weekly food consumption was recorded for each cage of non-recovery adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum. Weekly food consumptions were performed weekly for each cage of recovery adults throughout the study period.

Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively for males throughout the study period and for females during maturation and the first two weeks of gestation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the final week of gestation and during lactation.

-Water Consumption
Water intake was measured gravimetrically throughout the study period, via the daily weighing of water bottles.

Oestrous cyclicity (parental animals):

A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

All animals mated within four days of pairing.

Litter observations:

Litter Data
On completion of parturition (Day 0 of post partum), the number of live and dead offspring was recorded.
For each litter the following was recorded:
-Number of offspring born
-Number and sex of offspring alive recorded daily and reported on Day 1 and 4 post partum
-Clinical condition of offspring from birth to Day 4 post partum
-Individual offspring and litter weights on Day 1 and 4 post partum

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):

Adult non-recovery males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult non-recovery females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.

In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted.

Recovery animals were killed by intravenous overdose of sodium pentobarbitone followed by exanguination fourteen days after the termination of treatment (Day 57).

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

- Organ Weights
The following organs, removed from the five selected males and parental females from each group that were killed at the end of the study, were dissected free from fat and weighed before fixation.:
Adrenals Liver
Brain Spleen
Heart Kidneys
Thymus

The reproductive organs shown below were weighed from all animals that were killed at the end of the study:
Ovaries
Epididymides
Testes

- Histopathology
Samples of the following tissues were preserved from five males and five females from each dose group, in buffered 10% formalin.
Adrenals Mammary gland
Aorta (thoracic) Muscle (skeletal)
Bone & bone marrow (femur including stifle joint) Pancreas
Bone & bone marrow (sternum) Oesophagus
Brain (including cerebrum, cerebellum and pons) Rectum
Caecum Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Skin (hind limb)
Eyes Spinal cord (cervical)
Gross lesions Spleen
Heart Stomach
Ileum Thyroid/parathyroid
Jejunum Trachea
Kidneys Thymus
Liver Urinary bladder
Lungs (with bronchi) # Vagina
Lymph nodes (cervical and mesenteric)

# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

The tissues shown below were also removed from the remaining animals:
Coagulating gland Prostate
Epididymides* Seminal vesicles
Ovaries Testes *
Pituitary Uterus/Cervix

* = preserved in Bouin’s fluid and then in 70% IMS after forty-eight hours

All tissues were despatched to Propath UK Ltd (Principal Investigator: N Candy). The tissues from five selected control and 1000 mg/kg/day dose group animals and those animals dying during the study, were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues from the remaining control and 1000 mg/kg/day were also processed.

Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of kidney from five animals per sex in the low and intermediate groups and all recovery group animals.

Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.

Postmortem examinations (offspring):

All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

The following parameters were subjected to statistical analysis:
Bodyweight and bodyweight change
Food consumption during gestation and lactation
Litter data
Implantation losses and viability indices
Offspring bodyweight and bodyweight change
Offspring surface righting
Adult absolute and bodyweight relative organ weights

The following statistical procedures were used:
Data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.
Non-parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
Probability values (p) are presented as follows:
p < 0.001***; p < 0.01**; p < 0.05*; P≥ 0.05 (not significant)

Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:
-Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
-Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.

Reproductive indices:

Reproductive Indices
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation.
-Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
-Fertility Indices
For each group the following were calculated:

Mating Index (%) = (Number of animals mated / Number of animals paired) x 100

Pregnancy Index (%) = (Number of pregnant females / Number of animals mated) x 100

Gestation and Parturition Data
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
-Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
-Parturition Index
The following was calculated for each group:

Parturition Index (%) = (Number of females delivering live offspring / Number of pregnant females) x 100

Offspring viability indices:

Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
-Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:

% pre-implantation loss = [(Number of Corpora Lutea - Number of implantation sites) / Number of corpora lutea] x 100

% post-implantation loss = [(Number of implantation sites - number of offspring) / Number of implantation sites] x 100


-Live Birth and Viability Indices
The following indices were calculated for each litter as follows:

Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) x 100

Viability Index 1 (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) x 100


-Sex Ratio (% males)
Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:
(Number of male offspring / Number of offspring of determined sex) x 100

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See below

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: See below

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Haematology: Reductions in haemoglobin, haematocrit and erythrocyte count were detected for 1000 mg/kg/day males at Day 14 of the study with a concomitant reduction in mean cell haemoglobin and mean cell volume. The reduction in haematocrit, mean cell haemoglobin and mean cell volume was also detected for 250 mg/kg/day males at Day 14. No such observations were detected for treated females during the Day 14 haematological analysis.
Haematological analysis carried out for non-recovery dose groups at termination (Day 43 for males and Day 5 post partum for females) revealed reductions in haemoglobin, mean cell haemoglobin (MCH) and mean cell volume (MCV) for animals of either sex treated with 1000 mg/kg/day. In addition females of this treatment group showed reductions in haematocrit, and the reduction in MCV and MCH extended into the male 250 mg/kg/day dose group. Females treated with 1000 mg/kg/day also showed reductions in haemoglobin and haematocrit after the fourteen day treatment free period.

Blood Chemistry: Females treated with 1000 or 250 mg/kg/day showed elevations in plasma urea during blood chemical analysis carried out at Day 14. Females treated with 1000 mg/kg/day continued to show the elevation in plasma urea at termination.

Mating performance, fertility and gestation length: There were no treatment-related effects on mating performance, fertility or gestation lengths.

Necropsy: findings among adult animals and their offspring did not indicate any effect of treatment at dosages of 50, 250 or 1000 mg/kg/day.

Organ weights: Females treated with 1000 mg/kg/day had elevated absolute kidney weights, whilst these females, 1000 mg/kg/day males and females treated with 250 mg/kg/day had elevated kidney weight relative to bodyweight. Elevations in absolute kidney weight were also detected for animals of either sex treated with 1000 mg/kg/day after the fourteen day treatment free period. Females treated with 1000 mg/kg/day also had elevated kidney weight relative to terminal bodyweight.

Histopathology: The following treatment related effects were detected:
KIDNEY: A greater incidence and generally higher grades of severity of groups of basophilic tubules, the presence of tubular dilatation, hypertrophy of the epithelium of collecting ducts, and hyperplasia of the renal pelvic/papillary epithelium, were all observed in relation to treatment for animals of either sex treated with 1000 mg/kg/day. In addition, and for females only at this dose level, cortico-medullary mineralisation was observed and acute inflammatory cell foci were seen in the renal papilla. Similar changes but with lesser prevalence and generally higher grades of severity were seen for animals of either sex treated with 250 mg/kg/day but not at 50 mg/kg/day.
Hypertrophy of the collecting duct epithelium remained among Recovery 1000 mg/kg/day animals of either sex following an additional fourteen days without treatment. Other treatment-related changes had largely regressed although globular accumulations of eosinophilic material, similar in appearance to α-2 microglobulin deposits, were observed for four male rats in the Recovery 1000 mg/kg/day group but not among Recovery Control male rats. α-2 Microglobulin is exclusive to the kidney of male rats.

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: kidney, haematological and blood chemical changes

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:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Litter Responses: There were no adverse effects in litter size, offspring viability, growth or development.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects on reproduction evident at all dose levels tested.

Reproductive effects observed:

not specified

TABULAR SUMMARY REPORT OF EFFECTS ON REPRODUCTION/DEVELOPMENT

Observations		Dose Level (mg/kg/day)
		0 (Control)	50	250	1000
Mated pairs	n	10	10	10	10
Females showing evidence of copulation	n	10	10	10	10
Pregnant females	n	10	10	10	10
Conception Days 1-5	n	10	10	10	10
Gestation = 22	n	1	4	2	0
Gestation = 22.5 days	n	5	1	2	0
Gestation = 23 days	n	2	3	5	9
Gestation = 23.5 days	n	2	2	1	1
Dams with live young born	n	10	10	10	10
Dams with live young at Day 5 post partum	n	10	10	10	10
Corpora lutea/dam	x	15.5	16.9	16.6	16.4
Implants/dam	x	14.3	15.4	14.7	15.3
Live offspring/dam Day 1 post partum	x	12.9	14.5	13.4	13.1
Live offspring/dam at Day 4 post partum	x	12.9	14.1	13.3	13.0
Sex ratio: % males Day 1 post partum	x	50.6	48.4	48.9	48.4
Sex ratio: % males at Day 4 post partum	x	50.6	48.6	48.5	48.7
Litter weight (g) at Day 1 post partum	x	96.2	107.6	100.6	96.1
Litter weight (g) at Day 4 post partum	x	138.1	149.4	143.0	133.4
Male - offspring weight (g) at Day 1 post partum	x	7.7	7.7	7.7	7.5
Male - offspring weight (g) at Day 4 post partum	x	11.1	10.9	11.1	10.5
Female offspring weight at Day 1 Post Partum	x	7.3	7.2	7.5	7.0
Female offspring weight at Day 4 Post Partum	x	10.6	10.4	10.9	10.1
LOSS OF OFFSPRING/DAM
Pre-implantation (corpora lutea minus implantations)
0	n	5	2	2	2
1	n	1	2	2	5
2	n	2	4	2	3
3	n	1	0	3	0
4	n	1	1	1	0
Pre-natal (implantations minus live births)
0	n	3	3	4	3
1	n	3	3	2	7
2	n	2	3	3	0
3	n	1	0	1	0
4	n	1	0	0	0
Post natal (live births minus offspring alive on Day 4 post partum)
0	n	10	6	8	6
1	n	0	1	1	3
2	n	0	2	1	0
13	n	0	0	0	1

n= Number

x= Mean

Conclusions:

Under the conditions of the study the NOEL was determined to be 1000 mg/kg/day.

Executive summary:

No treatment-related effects on reproduction were evident for the females that had litters therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg/day. The study was conducted in accordance with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study was conducted under GLP to the standardised guidelines OECD 422 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test”.
In accordance with the criteria of Klimisch (1997) it was awarded a reliability score of 1.

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 effects of the test material on reproductive function in the Sprague-Dawley rat were investigated in accordance with GLP and the standardised guideline OECD 422.

In the sub-chronic study, the animals were exposed to dose levels of 50, 250 and 1000 mg/kg/day. No treatment-related effects on reproduction were evident for the females that had litters, therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg/day.

7.8.1 Toxicity to reproduction

In accordance with Section 1 of REACH Annex XI, the two generation reproductive toxicity study (OECD 416) does not appear scientifically necessary. The results of the OECD 422 study showed no concern for this endpoint and there are no other reasonable grounds for concern.

Short description of key information:
The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg/day.

Justification for selection of Effect on fertility via oral route:
Only one study available.

Effects on developmental toxicity

Description of key information

The ‘No Observed Effect Level’ (NOEL) for developmental toxicity/teratogenicity was considered to be 1000 mg/kg/day. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 October 2006 - 04 April 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

other: OECD 422

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Strain: Sprague-Dawley
- Source: Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: approximately 8 weeks.
- Weight at study initiation: males: 257 - 318 g; females: 198 - 255 g.
- Housing: Initially, all animals were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. During the mating phase for the non-recovery dose groups, animals were transferred to similar cages on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes (Datesand Ltd. Cheshire, UK). Recovery animals were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper.
- Diet (e.g. ad libitum): ad libitum. A pelleted diet (Rodent PMI 5002 (Certified) diet, BCM IPS Limited, London, UK) was used throughout the study.
- Water (e.g. ad libitum): ad libitum. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 14 days.

The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
Environmental enrichment was provided in the form of wooden chew blocks (B & K Universal Ltd, Hull, UK) and cardboard fun tunnels (Datesand Ltd, Cheshire, UK) except for mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 15% relative
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

The stability and homogeneity of the test material formulations were determined analytically and show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately at 4°C in the dark
The vehicle was chosen due to the complex nature of the test material and its limited solubility in organic and aqueous media.

Treatment Group Dose Level Treatment Volume Concentration
(mg/kg/day) (mL/kg) (mg/mL)
Control 0 4 0
Low 50 4 12.5
Intermediate 250 4 62.5
High 1000 4 250
Recovery Control 0 4 0
Recovery High 1000 4 250

The test material was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg/day of Arachis oil BP.

The volume of test and control material administered to each animal was based on the most recent scheduled bodyweight and was adjusted at regular intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical Analysis of Test Material Formulations, Methods and Results
METHOD OF ANALYSIS
Due to the complex nature of the test material and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed. The concentration of 12-Aminododecanoic acid in the test material formulations was determined using a gravimetric technique.

-Samples
The test material formulations were weighed into tared glass sintered crucibles and then rinsed with acetone to leave a test material residue. The samples were then dried in an oven at approximately 105 °C before being allowed to cool over silica gel in a desiccator and re-weighed.
-Homogeneity Determinations
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.
-Stability Determinations
The test material formulations were sampled and analysed initially and then after storage at approximately +4 °C in the dark for fourteen days.
The analytical procedure is considered to be non stability indicating, however the residue was subjected to an FTIR scan and the results suggest the test material is stable in the formulation.
-Verification of Test Material Formulation Concentrations
The test material formulations were sampled and analysed within two days of preparation

Homogeneity of Test Material Formulations:

Nominal Concentration Sampling Location Concentration Found (mg/mL)
(mg/mL) 1 2 3 Mean
Top 13.2 12.7 13.4 13.1
12.5 Middle 11.5 13.1 13.2 12.6
Bottom 13.5 13.1 13.4 13.3
Top 250 245 246 247
250 Middle 246 242 236 241
Bottom 248 235 224 236


Stability of Test Material Formulations:

Nominal Concentration Concentration Found Initially Concentration Found After Storage for Fourteen Days
(mg/mL) (mg/mL) (mg/mL) (expressed as % of initial)
12.5 13.0 11.5 88
250 241 248 103


Verification of Concentration of Weekly Test Material Formulation:

Week No. Nominal Concentration Concentration Found
(mg/mL) (mg/mL) (expressed as % of nominal)
0 ND -
12.5 12.1 97
1 62.5 58.8 94
250 243 97

0 ND -
12.5 12.7 101
2 62.5 61.1 98
250 246 98

0 ND -
12.5 12.7 99
3 62.5 62.0 99
250 245 98

0 ND -
12.5 12.9 103
4 62.5 65.3 104
250 248 99

0 ND -
12.5 12.5 100
5 62.5 61.0 98
250 246 98

0 ND -
12.5 13.3 107
6 62.5 64.0 102
250 246 98

0 ND -
12.5 10.8 87
7 62.5 61.2 98
250 241 96

ND = none detected
- = not applicable

METHOD VALIDATION
Specificity
A blank Arachis Oil BP (control) was analysed. The analysis of the blank Arachis Oil BP (control) produced no signal that interfered with the signal due to the test material.

Accuracy

Samples of Arachis Oil BP were accurately fortified with known amounts of test material, and analysed:
Fortification Concentration Found Recovered Mean Recovery
(mg/g) (mg/g) (%) (%)
12.6 12.9 103 103
12.9 13.6 104
244 234 96 97
244 239 98
The analytical method has been considered to be sufficiently accurate for the purpose of this study. The test sample results have not been corrected for recovery.

Conclusion
The analytical method has been satisfactorily validated in terms of specificity and accuracy for the purposes of the study.

Details on mating procedure:

Initially, all animals were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. During the mating phase for the non-recovery dose groups, animals were transferred to similar cages on a one male: one female basis. Following evidence of mating using vaginal smearing, the males were returned to their original cages and females were transferred to individual cages.

Non-recovery animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Duration of treatment / exposure:

54 days (treatment groups), 42 days (recovery groups).

Frequency of treatment:

Daily

Duration of test:

Up to 54 days (recovery group = 42 days)

Remarks:

Doses / Concentrations:
0, 50, 250 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex per dose in the treatment groups.
5 animals per sex per dose in the recovery groups.

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were chosen based on the results of a preliminary range-finder.

The test material was administered by gavage to three groups each of ten male and ten female rats for up to fifty-four consecutive days, at dose levels of 50, 250 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for forty-two consecutive days and then maintained without treatment for a further fourteen days.
Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating and at termination on five selected non-recovery males and females from each dose group. In addition, haematology and blood chemistry were evaluated after the fourteen day treatment free period on all recovery animals. Urinalysis was evaluated on five selected non-recovery males at the end of the treatment period and for all recovery group males at the end of the treatment free period.

Maternal examinations:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before and after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). During the treatment-free period, recovery animals were observed twice daily (once daily at weekends). All observations were recorded.

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females per non-recovery dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose-built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin colour
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behaviour Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation

Functional Performance Tests
-Motor Activity: Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period).

-Forelimb/Hindlimb Grips Strength: An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

- Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli:
Grasp response Touch escape
Vocalisation Pupil reflex
Toe pinch Startle reflex
Tail pinch Blink reflex
Finger approach

-Bodyweight
Individual bodyweights were recorded on Day 0 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination.

-Food Consumption
During the maturation period, weekly food consumption was recorded for each cage of non-recovery adults. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum. Weekly food consumptions were performed weekly for each cage of recovery adults throughout the study period.

Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively for females during maturation and the first two weeks of gestation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the final week of gestation and during lactation.

-Water Consumption
Water intake was measured gravimetrically throughout the study period, via the daily weighing of water bottles.

Ovaries and uterine content:

The corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted.

Fetal examinations:

Litter Data
On completion of parturition (Day 0 of post partum), the number of live and dead offspring was recorded.
For each litter the following was recorded:
-Number of offspring born
-Number and sex of offspring alive recorded daily and reported on Day 1 and 4 post partum
-Clinical condition of offspring from birth to Day 4 post partum
-Individual offspring and litter weights on Day 1 and 4 post partum

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Statistics:

The following parameters were subjected to statistical analysis:
Bodyweight and bodyweight change
Food consumption during gestation and lactation
Litter data
Implantation losses and viability indices
Offspring bodyweight and bodyweight change
Offspring surface righting
Adult absolute and bodyweight relative organ weights

The following statistical procedures were used:
Data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.
Non-parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
Probability values (p) are presented as follows:
p < 0.001***; p < 0.01**; p < 0.05*; P≥ 0.05 (not significant)

Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:
-Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
-Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.

Indices:

-Fertility Indices
For each group the following were calculated:

Mating Index (%) = (Number of animals mated / Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females / Number of animals mated) x 100

Gestation and Parturition Data
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
-Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
-Parturition Index
The following was calculated for each group:

Parturition Index (%) = (Number of females delivering live offspring / Number of pregnant females) x 100

Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
-Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:

% pre-implantation loss = [(Number of Corpora Lutea - Number of implantati on sites) / Number of corpora lutea] x 100
% post-implantation loss = [(Number of implantation sites - number of offspring) / Number of implantation sites] x 100


-Live Birth and Viability Indices
The following indices were calculated for each litter as follows:

Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) x 100
Viability Index 1 (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) x 100


-Sex Ratio (% males)
Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:
(Number of male offspring / Number of offspring of determined sex) x 100

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
The physical health of the parental animals was not impaired and daily clinical observations, weekly open field arena observations and functional performance tests did not show any effects attributed to toxicity. Bodyweight change and dietary intake were not affected for animals of either sex throughout the study.

Haematological assessments at termination for animals of either sex treated with 1000 mg/kg/day revealed a slight anaemic response to treatment. This was characterised by reductions detected in haemoglobin, haematocrit, erythrocyte count which resulted in reductions in mean cell haemoglobin and mean cell volume. Reductions in haemoglobin and haematocrit were also detected for females treated with 1000 mg/kg/day after the fourteen day treatment free period. Although a microscopic effect of toxicity on the haemopoetic system has not been identified during histopathological examinations, the reduction in the haematological parameters identified cannot be disregarded and are considered to represent an adverse health effect.

Elevations in water consumption were detected throughout the treatment period for animals of either sex treated with 1000 or 250 mg/kg/day. In addition elevations in plasma urea were detected for females treated with 1000 or 250 mg/kg/day during blood analysis carried out at Day 14; and for 1000 mg/kg/day females at termination. These findings suggest impaired function of the kidneys. This is supported by elevated kidney weights and microscopic findings for animals of either sex treated with 1000 or 250 mg/kg/day; these included, a greater incidence and higher grades of severity of groups of basophilic tubules, the presence of tubular dilatation, hypertrophy of the epithelium of collecting ducts, and hyperplasia of the renal pelvic/papillary epithelium. In addition, cortico-medullary mineralisation was observed and acute inflammatory cell foci were seen in the renal papilla for females treated with 1000 mg/kg/day. Similar changes but with lesser prevalence and general severity were seen for animals of either sex treated with 250 mg/kg/day but not at 50 mg/kg/day.

Hypertrophy of the collecting duct epithelium remained among animals of either sex treated with 1000 mg/kg/day following the additional fourteen days without treatment.

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No treatment-related effects on reproduction for the females that had litters were evident.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

TABULAR SUMMARY REPORT OF EFFECTS ON REPRODUCTION/DEVELOPMENT

Observations		Dose Level (mg/kg/day)
		0 (Control)	50	250	1000
Mated pairs	n	10	10	10	10
Females showing evidence of copulation	n	10	10	10	10
Pregnant females	n	10	10	10	10
Conception Days 1-5	n	10	10	10	10
Gestation = 22	n	1	4	2	0
Gestation = 22.5 days	n	5	1	2	0
Gestation = 23 days	n	2	3	5	9
Gestation = 23.5 days	n	2	2	1	1
Dams with live young born	n	10	10	10	10
Dams with live young at Day 5 post partum	n	10	10	10	10
Corpora lutea/dam	x	15.5	16.9	16.6	16.4
Implants/dam	x	14.3	15.4	14.7	15.3
Live offspring/dam Day 1 post partum	x	12.9	14.5	13.4	13.1
Live offspring/dam at Day 4 post partum	x	12.9	14.1	13.3	13.0
Sex ratio: % males Day 1 post partum	x	50.6	48.4	48.9	48.4
Sex ratio: % males at Day 4 post partum	x	50.6	48.6	48.5	48.7
Litter weight (g) at Day 1 post partum	x	96.2	107.6	100.6	96.1
Litter weight (g) at Day 4 post partum	x	138.1	149.4	143.0	133.4
Male - offspring weight (g) at Day 1 post partum	x	7.7	7.7	7.7	7.5
Male - offspring weight (g) at Day 4 post partum	x	11.1	10.9	11.1	10.5
Female offspring weight at Day 1 Post Partum	x	7.3	7.2	7.5	7.0
Female offspring weight at Day 4 Post Partum	x	10.6	10.4	10.9	10.1
LOSS OF OFFSPRING/DAM
Pre-implantation (corpora lutea minus implantations)
0	n	5	2	2	2
1	n	1	2	2	5
2	n	2	4	2	3
3	n	1	0	3	0
4	n	1	1	1	0
Pre-natal (implantations minus live births)
0	n	3	3	4	3
1	n	3	3	2	7
2	n	2	3	3	0
3	n	1	0	1	0
4	n	1	0	0	0
Post natal (live births minus offspring alive on Day 4 post partum)
0	n	10	6	8	6
1	n	0	1	1	3
2	n	0	2	1	0
13	n	0	0	0	1

n= Number

x= Mean

Conclusions:

Under the conditions of the study the NOEL was determined to be 1000 mg/kg/day.

Executive summary:

No treatment-related effects on reproduction were evident for offspring, therefore the ‘No Observed Effect Level’ (NOEL) for developmental toxicity was considered to be 1000 mg/kg/day. The study was conducted in accordance with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study was conducted under GLP to the standardised guidelines OECD 422 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test”.
In accordance with the criteria of Klimisch (1997) it was awarded a reliability score of 1.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The effects of the test material on developmental parameters in the Sprague-Dawley rat were investigated in accordance with GLP and the standardised guideline OECD 422.

In the sub-chronic study, the animals were exposed to dose levels of 50, 250 and 1000 mg/kg/day. No treatment-related effects on offspring were evident, therefore the ‘No Observed Effect Level’ (NOEL) for developmental toxicity was considered to be 1000 mg/kg/day.

7.8.2 Developmental toxicity/teratogenicity

In accordance with Section 1 of REACH Annex XI, the prenatal developmental toxicity study (OECD 414) does not appear scientifically necessary. The results of the OECD 422 study showed no concern for this endpoint and there are no other reasonable grounds for concern.

Justification for selection of Effect on developmental toxicity: via oral route:
Only one study available.

Justification for classification or non-classification

In accordance with the criteria set out in Annex I of Regulation No. 1272/2008 the test material does not require classification for toxicity to reproduction. There is no evidence of adverse effects on sexual function and fertility or on development of offspring, caused by exposure.