Quaternary ammonium compounds, C18-22 (even numbered) alkyltrimethyl, chlorides; Docosyltrimethylammonium chloride; C18-22 TMAC

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the results of the read across study, the test substance is not considered to pose reproductive or development concern at the tested dose levels. The NOAELs for reproductive and development toxicity are considered to be at 75 and 30 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From October 20, 2009 to May 28, 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Animals: Rat, HanRcc: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
Number of Animals: 40 males (10 per group) and 40 females (10 per group)
Age (at Start of Treatment): 11 weeks
Body Weight Range (at Start of Treatment): Males (286 to 330 g) and females (181 to 205 g)
Identification: Cage card and individual animal number (ear tattoo).
Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation were taken into consideration in order to ensure similar mean body weights in all groups.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
Diet: Pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no. 60/09).
Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

DOSE FORMULATIONS

The dose formulations were prepared weekly using the test substance as supplied by the Sponsor.

C20/22 ATQ trocken was weighed into a plastic dish on a tared precision balance, transferred in a brown glass and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.


STORAGE OF DOSE FORMULATIONS

Dose formulations were stored at room temperature (20 ± 5 °C) in brown glass beakers.

Based upon the results of stability analyses performed within the Harlan Laboratories study C40763, non-GLP, dose formulations were stable for at least one week.


TREATMENT

Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
Frequency of Administration: Once daily
Target Dose Levels: Group 1: 0 mg/kg/day (control group); Group 2: 10 mg/kg/day; Group 3: 30 mg/kg/day; Group 4: 75 mg/kg/day
Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study C40763, where the administration of the test item was by gavage and by diet. The dose levels used where 50 and 150 mg/kg/ day as well as 600, 1800 and 6000 ppm. The groups receiving 150 mg/kg/day, 1800 or 6000 ppm had to be sacrificed for ethical reasons before the planned study termination due to marked body weight loss and general bad conditions. The NOAEL was considered to be 50 mg/kg/day or 600 ppm.
Dose Volume: 10 mL/kg body weight
Duration of Acclimatization Period: 9 days
Duration of Treatment Period: Males (minimum 4 weeks); females (approximately 7 weeks)

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day of mating was designated day 0 post coitum.
All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DOSE FORMULATIONS

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (4 hrs and 6 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Analytical Department (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by HPLC coupled to an ELSD detector following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

The application formulations investigated during the study were found to comprise C20/22-ATQ trocken in the range of 94.5% to 106.9% and, thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of C20/22-ATQ trocken in the preparations was approved because single results found did not deviate more than 4.2% (<15%) from the corresponding mean. In addition, the test item was found to be stable in application formulations when kept 6 days at room temperature (20±5°C) due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

Duration of treatment / exposure:

Males: Minimum 4 weeks
Females: Approximately 7 weeks

Frequency of treatment:

Once daily

Details on study schedule:

Acclimatization: 9 days (males and females)
First Test substance Administration : Day 1 of pre-pairing (males and females)
Pre-Pairing: 14 days (males and females)
Pairing: 14 days maximum (males and females)
Gestation: Approximately 21 days (females)
Treatment Ends: On day before sacrifice (males); on day 3 post partum (females)
Necropsy: After a minimum of 28 days treatment (males); on day 4 post partum (females)

Dose / conc.:

0 other: mg/kg bw/day (actual ingested)

Dose / conc.:

10 other: mg/kg bw/day (actual ingested)

Dose / conc.:

30 other: mg/kg bw/day (actual ingested)

Dose / conc.:

75 other: mg/kg bw/day (actual ingested)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.
Food Consumption: Males (weekly during pre-pairing period); females (pre-pairing period days 1 - 8 and 8 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and days 1 - 4 post partum). No food consumption was recorded during the pairing period.
Body Weights: Recorded daily from treatment start to day of necropsy.

Litter observations:

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

Postmortem examinations (parental animals):

TERMINATION OF THE STUDY

Males were sacrificed after they had been treated for at least 28 days. Dams were sacrificed on day 4 post partum.

If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.


NECROPSY

At the scheduled sacrifice, all parent animals were killed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes at the scheduled necropsy.

For the parent animals, special attention was directed at the organs of the reproductive system.
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.


ORGAN WEIGHTS

At the scheduled sacrifice, the testes and epididymides of all parental males were weighed as pairs.


TISSUE PRESERVATION

The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.

The testes and epididymides from all parental males were preserved in Bouin’s fixative. The prostate and seminal vesicles from all males were fixed in neutral phosphate buffered 4% formaldehyde solution.


HISTOTECHNIQUE

All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin.


HISTOPATHOLOGY

Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made.

A peer review was carried out by the Department of Pathology, Harlan Laboratories Ltd, Itingen / Switzerland.

Postmortem examinations (offspring):

TERMINATION OF THE STUDY

Pups were sacrificed on day 4 post partum.


NECROPSY

At the scheduled sacrifice, all pups were killed by an injection of sodium pentobarbital.

Dead pups, except those excessively cannibalized, were examined macroscopically.

All pups were examined macroscopically for any structural changes at the scheduled necropsy.

Statistics:

The following statistical methods were used to analyze food consumption, body weights, reproduction data, organ weights and macroscopic findings:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size.

Offspring viability indices:

From the on-line recorded reproduction data, the following parameters were calculated: dead/live pups at first litter check, pup sex ratios and postnatal loss (up to day 4 post partum).

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males at 75 and 30 mg/kg/day and in females at 75 mg/kg bw/day food consumption was decreased. In males and females at the dose level of 75 mg/kg bw/day body weights and body weight gain were decreased.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males at 75 and 30 mg/kg/day and in females at 75 mg/kg bw/day food consumption was decreased. In males and females at the dose level of 75 mg/kg bw/day body weights and body weight gain were decreased.

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:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

by means of testis and epididymis weights, histopathological examination of of testes, epididymides, prostate and seminal vesicles with special emphasis on the stages of spermatogenesis and histopathology of interstitial cell structure.

Reproductive performance:

no effects observed

1 IN-LIVE DATA - PARENTAL ANIMALS

1.1 CLINICAL SIGNS OR OBSERVATIONS
All animals survived until the scheduled necropsy.
In group 4, ruffled fur was noted in female no. 72 on day 22 of the gestation until day 2 of lactation, in female no. 73 on day 17 of gestation and in female no. 75 on days 2, 3 and 4 of lactation period. Vaginal bleeding was observed in female no. 79 on days 16 and 17 of gestation. These clinical signs were considered to be incidental since they were occasionally noted in single females.

1.2 FOOD CONSUMPTION OF MALES
Pre-pairing Period:
In groups 3 and 4, mean food consumption was dose-dependently and statistically significantly decreased during the entire pre-pairing period (-11.0% and -19.4% compared to the control, respectively). This was considered to be due to the treatment with the test item.
In group 2, no test item-related effects were noted.

1.3 FOOD CONSUMPTION OF FEMALES
Pre-pairing, Gestation and Lactation Periods:
In group 4, during the pre-pairing period mean food consumption was lower during the first week (-12.0% compared to the control). During the gestation period, mean food consumption started to decrease on days 7 - 14 (-7.4% compared to the control) followed by a marked decrease on days 14 - 21 (-17.8% compared to the control) through the lactation period (-20.2% compared to the control). These reductions were considered to be test item-related although they only attained statistical significance on the first week of the pre-pairing and during the last week of gestation period.
In groups 2 and 3, no test item-related effects were noted. The lower differences which occurred occasionally in group 3 were considered to be incidental.

1.4 BODY WEIGHTS OF MALES
Pre-pairing and Pairing Periods:
In group 4, during the pre-pairing period mean body weight was statistically significantly lower starting on day 7 onwards and mean body weight gain was statistically significantly lower during the entire pre-pairing period (weight gain: +3.3% versus +10.1%). This was considered to be a test item-related effect. During the pairing period, mean body weight remained statistically significantly lower while mean body weight gain was similar to the control (weight gain: +8.8% versus +10.5%).
In groups 2 and 3, no test item-related effects were observed in body weight and body weight gain.

1.5 BODY WEIGHTS OF FEMALES
Pre-pairing, Gestation and Lactation Periods:
In group 4, during the pre-pairing period mean body weight gain was statistically significantly decreased starting on day 9 until the end of the period while mean body weight was not affected (overall weight gain 4.7% versus +6.8%). During the gestation period, mean body weight was statistically significantly lower between days 16 and 21 and mean body weight gain was statistically significantly lower at the end of the period (overall weight gain +44.9% versus +56.6%). During the lactation period, mean body weight was statistically significantly lower for the whole period while mean body weight gain was not affected.
In groups 2 and 3, no test item-related effects were noted.

1.6 MATING PERFORMANCE AND FERTILITY
The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 1.9, 3.0, 2.6 and 2.1 days in order of ascending dose level. The median precoital time was 2, 3, 3 and 2 days in order of ascending dose level. In group 4, one female mated on the first day on the second pairing period.
One female (no. 42) in group 1 was not pregnant. Thus the fertility indices were 90.0% in group 1 and 100.0% in groups 2, 3 and 4.

1.7 DURATION OF GESTATION
The mean duration of gestation was unaffected by treatment with the test item. Mean duration of gestation was 21.4, 21.4, 21.6 and 21.6 days, in order of ascending dose level.

1.8 CORPORA LUTEA COUNT
The mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (13.9, 13.7, 13.3 and 13.3 in order of ascending dose level) and gave no indication of any test item-related effect.

1.9 IMPLANTATION RATE AND POST-IMPLANTATION LOSS
The mean number of implantations per dam was not affected by the treatment with the test item. The mean numbers of implantations per litter were 13.4, 13.5, 11.4 and 12.2 in order of ascending dose level. In group 3, the lower mean number of implantations was considered to be incidental since it did not follow a dose-dependent pattern and the value in group 4 was within the range of the historical control data.
Mean incidence of post-implantation loss as a percentage of total implantations was 8.3, 8.1, 12.3 and 17.3% in order of ascending dose level. In group 4, total number of post-implantation losses was statististically significantly higher. This was due to one dam (no. 72) which had total post-implantation loss (three post-implantation losses and ten pups found dead at first litter check). Since this occurred in one single female, post-implantation loss was not considered to be affected by the treatment with the test item.

1.10 LITTER SIZE AT FIRST LITTER CHECK
The number of live pups at first litter check was unaffected by treatment with the test item. The mean number of live pups per litter was 12.3, 12.4, 10.0 and 10.1 in order of ascending dose level and within the range of the historical control data. One dam (no. 72) in group 4 cannibalized all its ten pups before first litter check.
Birth index was 91.7, 91.9, 87.7 and 82.7% in order of ascending dose level. The statistically significantly lower birth index in group 4, which was due to whole litter loss of one single animal (no. 72) was still within the range of the historical control data.

1.11 POSTNATAL LOSS DAYS 0 - 4 POST PARTUM
In group 4, total postnatal loss was statistically significantly higher (seven pups versus 0 in the control). This was due to one dam (no. 75), which did not consume any food during the lactation period and which lost five pups on day 4 post partum. The postnatal loss was within the range of the historical control data and therefore not considered to be test item-related.
For females, which reared their pups until day 4 post partum, mean litter size on day 4 post partum was 12.3, 12.4, 10.0 and 10.5 pups in groups 1, 2, 3 and 4, respectively. Lower litter size in group 3 was secondary to the lower number of implantation sites.
The resulting viability indices were 100.0%, 100.0%, 100.0% and 92.3% in order of ascending dose levels. The statistically significantly lower viability index in group 4 was within the range of the historical control data.


2 TERMINAL FINDINGS - PARENTAL ANIMALS

2.1 ORGAN WEIGHTS
Absolue and relative weight of testes and epididymides were not affected by the treatment with the test item.

2.2 MACROSCOPICAL FINDINGS
In group 4, size of both testes and epdidimides were reduced in one male. One female was noted to have both uterine horns discolored dark-red and with firm reddish nodules in the mucosa of the right horn. In group 3, one female had left uterine horn dilated with mucous. Since these findings occurred isolated in single animals, they were not considered to be test item-related.

2.3 HISTOPATHOLOGY FINDINGS
Marked tubular atrophy of the testes and massive hypospermia of the epididymides were recorded in one male (no. 39) of group 4. These findings were considered to be spontaneous changes and not to be test item-related lesions, because minimal tubular atrophy of the testes was also recorded in one male of the control group and there were neither further findings of testes toxicity nor sperm staging abnormality in other males of group 4. All other findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age. No test item-related histological finding was recorded in ovary from two females (no. 42, 79) that did not give birth.

Sperm staging:
No differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

for parental toxicity

Effect level:

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

Sex:

male/female

Basis for effect level:

other: based on the decreased food consumption and lower body weights in males and females, reduced body weight gain in females at 75 mg/kg/day

Remarks on result:

other: Generation: P (parental toxicity)

Key result

Dose descriptor:

NOAEL

Remarks:

for reproductive toxicity

Effect level:

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

Sex:

male/female

Basis for effect level:

other: due to absence of effects on reproduction (fertility)

Remarks on result:

other: Generation: P (reproduction)

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 75 mg/kg/day, pup weight gain and pup weight on day 4 post partum were reduced. This effect was considered to be adverse.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

1 IN-LIVE DATA - F1 PUPS

1.1 EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION
In group 4, all pups in litter no. 72 were found dead. All these pups were cannibalized and/or in advanced autolysis. No other external findings were noted, therefore these findings were considered to be incidental.

1.2 SEX RATIOS
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item.
The proportion of males on day 4 post partum was 54%, 51%, 42% and 44% in order of ascending dose level.

1.3 PUP WEIGHTS TO DAY 4 POST PARTUM
The mean pup weights were were similar to the control group. On day 1 post partum mean pup weights were 5.7, 5.9, 5.9 and 5.5 g for combined data of male and female pups in order of ascending dose level.
In group 4, mean pup weight gain up to day 4 postpartum was lower than the control group (+29.1% versus +38.6%) and the mean pup weight was lower on day 4 postpartum (7.1 g versus 7.9 g). These differences were not statistically significant and most likely due to nursing problems based on lower food consumption of the group 4 females during lactation. In groups 2 and 3, mean pup weight gain up to day 4 post partum were unaffected by the treatment with the test item. Mean pup weights on day 4 postpartum were 8.3 and 8.4 g compared to 7.9 g in the control group for combined data of male and female pups.

1.4 MACROSCOPICAL FINDINGS
No relevant abnormal findings were noted at macroscopic examination of the pups. In group 4, all pups in one litter which were found dead at first litter check were cannibalized and/or advanced autolysed. No other findings were noted.

Key result

Dose descriptor:

NOAEL

Remarks:

for development toxicity

Generation:

F1

Effect level:

ca. 30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on the lower pup weight gain at 75 mg/kg/day, which was probably secondary to lower food consumption of the dams.

Remarks on result:

other: Generation: F1 (development)

Key result

Reproductive effects observed:

no

P Animals Breeding for F1 Litters

Group (mg/kg/day)	1 (0)	2 (10)	3 (30)	4 (75)
Female numbers	41-50	51-60	61-70	71-80
Number of females paired	10	10	10	10
Number of females mated	10	10	10	10
Number of pregnant females (A)	9	10	10	10
Number of females with only implantation sites (B)				1
Numbers of females which cannibalized the whole litter (C)				1
Number of females which reared their pups until day 4 post partum	9	10	10	8

(A)  Female No. 42 in group 1 was not pregnant.

(B)  Female No. 79 in group 4 had only implantation sites.

(C)   Female No. 72 in group 4 had cannibalized the whole litter at first litter check.

Conclusions:

Under the study conditions, based on the decreased food consumption and lower body weights in males and females or reduced body weight gain in females at 75 mg/kg/day, the NOAEL for parental toxicity was established at 30 mg/kg bw/day. The NOAEL for reproduction (fertility) due to absence of toxicity was considered to be at 75 mg/kg bw/day (the highest tested dose), whereas, the NOAEL for developmental toxicity was considered to be 30 mg/kg bw/day, based on the lower pup weight gain at 75 mg/kg bw/day, which was probably secondary to lower food consumption of the dams.

Executive summary:

A screening study was conducted to determine the developmental toxicity of the read across substance, C20-22 TMAC (active: 97.8%), according to OECD Guideline 421, in compliance with GLP. In the study, four groups of 10 males and 10 females were treated once daily by gavage with the read across substance at dose levels of 0, 10, 30 and 75 mg/kg bw/day. A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (i.e., highly purified water). The males were treated over 14 d before pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to Day 4 post partum. In parental animals, all animals survived until the scheduled necropsy. No read across substance-related clinical signs were observed. In males at 30 and 75 mg/kg bw/day, mean food consumption was dose-dependently and statistically significantly decreased during the pre-pairing period. In females at 75 mg/kg bw/day, mean food consumption was decreased during the first week of pre-pairing period and starting from the second week of gestation until the end of the study. In males at 75 mg/kg bw/day, mean body weight was statistically significantly lower starting on Day 7 of the pre-pairing period and through the pairing period. In females at 75 mg/kg bw/day, mean body weight was statistically significantly lower between Days 16 and 21 of the gestation and during the lactation period. The mean body weight gain was statistically significantly lower on Days 9, 10 and 13 of the pre-pairing period and on the last day of the gestation. The mating performance, fertility, duration of gestation, number of corpora lutea and implantation sites were not affected by the treatment with the t read across substance. Taking into consideration that the total post-implantation loss occurred only in one dam at 75 mg/kg bw/day, the incidence of post-implantation losses was considered to be within the range of the historical control data and was not considered to be affected by the treatment with the read across substance. No read across substance-related effects were observed in absolute and relative weight of testes and epididymides. There was no macrocospical finding which was attributed to the treatment with the read across substance. Histopathological examination did not reaveal any read across substance-related alteration. No differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals. In F1 Pups, the number of live pups at first litter check and the mean litter size were unaffected by treatment with the read across substance. One dam lost its whole litter at first litter check. Since this occurred in one single dam, it was considered to be incidental. The resulting lower birth index at 75 mg/kg bw/day was still within the range of the historical control data. Postnatal loss was also considered to be unaffected since it was observed only at 75 mg/kg/day and in one dam, which did not consume any food during the lactation period and lost five pups. This was within the range of historical controls. Sex ratios at first litter check and on Day 4 post partum were not affected by treatment with the read across substance. At 75 mg/kg bw/day, pup weight gain and pup weight on Day 4 post partum were statically non-significantly reduced, which was most likely due to nursing problems based on lower food consumption of the females of this group during lactation. Under the study conditions, based on the decreased food consumption and lower body weights in males and females, reduced body weight gain in females at 75 mg/kg/day and the lower food consumption in males at 30 mg/kg/day, which did not affect body weight, the NOAEL for parental toxicity was established at 30 mg/kg bw/day. The NOAEL for reproduction (fertility) due to absence of toxicity was considered to be at 75 mg/kg bw/day (the highest tested dose), whereas, the NOAEL for developmental toxicity was considered to be 30 mg/kg bw/day, based on the lower pup weight gain at 75 mg/kg bw/day, which was probably secondary to lower food consumption of the dams (Ceccatelli, 2010). Based on the results of the read across study, similar NOAELs can be expected for the test substance, C18-22 TMAC.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline compliant screening study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A screening study was conducted to determine the developmental toxicity of the read across substance, C20-22 TMAC (active: 97.8%), according to OECD Guideline 421, in compliance with GLP. In the study, four groups of 10 males and 10 females were treated once daily by gavage with the read across substance at dose levels of 0, 10, 30 and 75 mg/kg bw/day. A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (i.e., highly purified water). The males were treated over 14 d before pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to Day 4 post partum. In parental animals, all animals survived until the scheduled necropsy. No read across substance-related clinical signs were observed. In males at 30 and 75 mg/kg bw/day, mean food consumption was dose-dependently and statistically significantly decreased during the pre-pairing period. In females at 75 mg/kg bw/day, mean food consumption was decreased during the first week of pre-pairing period and starting from the second week of gestation until the end of the study. In males at 75 mg/kg bw/day, mean body weight was statistically significantly lower starting on Day 7 of the pre-pairing period and through the pairing period. In females at 75 mg/kg bw/day, mean body weight was statistically significantly lower between Days 16 and 21 of the gestation and during the lactation period. The mean body weight gain was statistically significantly lower on Days 9, 10 and 13 of the pre-pairing period and on the last day of the gestation. The mating performance, fertility, duration of gestation, number of corpora lutea and implantation sites were not affected by the treatment with the t read across substance. Taking into consideration that the total post-implantation loss occurred only in one dam at 75 mg/kg bw/day, the incidence of post-implantation losses was considered to be within the range of the historical control data and was not considered to be affected by the treatment with the read across substance. No read across substance-related effects were observed in absolute and relative weight of testes and epididymides. There was no macrocospical finding which was attributed to the treatment with the read across substance. Histopathological examination did not reveal any read across substance-related alteration. No differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals. In F1 Pups, the number of live pups at first litter check and the mean litter size were unaffected by treatment with the read across substance. One dam lost its whole litter at first litter check. Since this occurred in one single dam, it was considered to be incidental. The resulting lower birth index at 75 mg/kg bw/day was still within the range of the historical control data. Postnatal loss was also considered to be unaffected since it was observed only at 75 mg/kg/day and in one dam, which did not consume any food during the lactation period and lost five pups. This was within the range of historical controls. Sex ratios at first litter check and on Day 4 post partum were not affected by treatment with the read across substance. At 75 mg/kg bw/day, pup weight gain and pup weight on Day 4 post partum were statically non-significantly reduced, which was most likely due to nursing problems based on lower food consumption of the females of this group during lactation. Under the study conditions, based on the decreased food consumption and lower body weights in males and females, reduced body weight gain in females at 75 mg/kg/day and the lower food consumption in males at 30 mg/kg/day, which did not affect body weight, the NOAEL for parental toxicity was established at 30 mg/kg bw/day. The NOAEL for reproduction (fertility) due to absence of toxicity was considered to be at 75 mg/kg bw/day (the highest tested dose), whereas, the NOAEL for developmental toxicity was considered to be 30 mg/kg bw/day, based on the lower pup weight gain at 75 mg/kg bw/day, which was probably secondary to lower food consumption of the dams (Ceccatelli, 2010). Based on the results of the read across study, similar NOAELs can be expected for the test substance, C18-22 TMAC.

Effects on developmental toxicity

Description of key information

See above discussion.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline compliant screening study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the absence of significant toxicity in the read across screening study, the test substance, C18-22 TMAC does not warrant classification for reproductive or development toxicity, according to EU CLP criteria (Regulation 1272/2008/EC).