Amines, C12-16 alkyl dimethyl, reaction products with ethyl chloroacetate, 2-(2-aminoethylamino)ethanol and 2-propenoic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

-study conducted according to OECD guideline 421 (adopted 29 July 2016), male and female Wistar rats were exposed to 0, 62.5, 125, 250 mg/kg bw /d aqueous solution of Cocobetainamidoamphopropionate (a.i. 50%) by oral gavage for 32 or 33 days and at least 51 days, respectively, no treatment related effects were observed up to the highest dose tested, NOAEL = 250 mg/kg bw/day (a.i. 50%)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of experimental phase First day of mating 07 September 2017 Allocation of animals to groups 17 August 2018 End of experimental phase Last day of necropsy for females 26 October 2017 Study completion Date of Study Director’s signature on this report

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Details of the test item received at RTC were as follows:
Identity Rewoteric QAM 50
Chemical name Aqueous solution of a Cocobetainamidoamphopropionate
Purity (solid content) 50.7%
Batch no. TS 83/17
Expiry date 28March 2018
Storage conditions Room temperature protected from light

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Hannover rat was the species and strain of choice because it is accepted by many regulatory authorities and because there are ample experience and background data on this species and strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of 102 Wistar Hannover rats (45 males and 57 virgin females) were ordered from Envigo RMS s.r.l., San Pietro al Natisone (UD), Italy. The animals were approximately 8 to 9 weeks old and weighing 221-230 g for males and approximately 176 to 185 g for females at receipt. Animalswere supplied by ENVIGO Kreuzelweg 53, 5961NM HorstNL 5960AD Horst P.O. Box 6174 (Netherlands). After arrival, the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian. An acclimatisation period of 22 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

The animalswere housed in a limited access rodent facility. Animal roomcontrolswere set to maintain temperature and relative humidity at 22°C±2°C and 55%±15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.

During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
After mating, the males were re-caged as they were before mating.
The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary and changed at least 2 times a week.

Drinking water was supplied ad libitum to each cage via water bottles.
A commercially available laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019, SettimoMilanese (MI), Italy) was offered ad libitum throughout the study.

Route of administration:

oral: gavage

Details on exposure:

The test item was administered orally, by gavage. The oral route was selected as it is a possible route of exposure of the test item in man.
The dose levels of 62.5, 125 and 250 mg/kg/day were selected by the Sponsor based on information from a previous study.

Details on mating procedure:

Pairing was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (spermidentification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in a separate study in order to validate both the analytical method and the formulation procedure and to verify the stability of the formulations (RTC Study no. A2749).
The analytical method was validated in the range from 10 to 50 mg/mL, using Metoprolol as internal standard. Linearity, accuracy and precision were within the limits stated in RTC SOPs for solutions (r > 0.98; accuracy 90-110%; precision CV <5%). In the same study, a 28 hour stability at room temperature was verified in the range from 10 to 50 mg/mL. The proposed formulation procedure for the test item was checked in the range from 10 to 50 mg/mL by chemical analysis (concentration) to confirmthat the method was suitable. All results for all levelswere within the acceptability limits stated in RTC SOPs for concentration (90-110%).
In the present study, samples of the formulations prepared on two occasions during the study (Week 1 and again towards the end of the study) were analysed to check the concentration. Chemical analysis was carried out by the Analytical Chemistry Department at RTC.
The software used for this activity was Analyst 1.6.2. Results of the analyses were within the acceptability limits stated in RTC SOPs for concentration of solutions (90-110%),

Duration of treatment / exposure:

Males
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the pairing period and thereafter until the day before necropsy (Days 33 and 34). Males were treated for a total of 32 or 33 days.
Dose volumes were adjusted once per week for each animal according to the last recordedbody weight.

Females
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum (for at least 51 days). One not pregnant female was dosed up to the day before necropsy. Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.

Frequency of treatment:

Animals were dosed once a day, 7 days a week

Dose / conc.:

62.5 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

125 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

250 mg/kg bw/day

Remarks:

Group 4

Dose / conc.:

0 mg/kg bw/day

Remarks:

Group 1 - Control group

No. of animals per sex per dose:

Each group comprised 10 male and 10 female rats.

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Mortality
Throughout the study, all animals were checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day.

Clinical signs
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions. All observations were recorded for individual animals.

Body weight
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy. Individual bodyweight data recorded for females during the mating period are not tabulated in this report but will be archived with all raw data.

Food consumption
The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.

Vaginal smears
Stock females
Oestrous cycle was monitored by vaginal smears in all stock females for at least 1 week before allocation, in order to exclude from the study females with irregular cycle. These data are not tabulated in this report, but will be archived with raw data.
Females allocated to groups
Vaginal smears were taken in the morning from Day 1 of treatment, up to positive identification of mating including 2 weeks before the pairing.
The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Vaginal smears were also taken from all females, before despatch to necropsy


Clinical pathology investigations

Parental animals
As a part of the necropsy procedure, approximately 0.8mLof blood sampleswere withdrawn under isofluorane anaesthesia from the abdominal vena cava from all parental male and female rats.
All blood samples mentioned above were transferred into tubes of appropriate capacity, containing no anticoagulant and centrifuged at roomtemperature. The serumobtained was divided in several aliquots for analysis as indicated in section 4.4.2. The aliquots obtained were stored at -80°C until analysis.

Bioanalysis - Thyroid hormone determination (T3, T4 and TSH)
Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by a multiplex assay, using LuminexMagpix system and the MILLIPLEX MAP Rat ThyroidMagnetic Bead Panel kit (MerkMillipore, cat. no. RTHYMAG-30K).
The determination was restricted as detailed below:
1. Samples from all parental males from all groups
2. Samples from pups on Day 14 post partum
The results of these analyses are presented as individual data, mean and standard deviation and reported in ng/mL.
Since no treatment related effects were seen in the determination performed in parental males and in pups on Day 14 post partum, samples obtained from females and from pups on Day 4 post partum were not analysed.

Oestrous cyclicity (parental animals):

Females allocated to groups
Vaginal smears were taken in the morning from Day 1 of treatment, up to positive identification
of mating including 2 weeks before the pairing.
The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of
mating)
Vaginal smears were also taken from all females, before despatch to necropsy.

Sperm parameters (parental animals):

Spermatogenic cycle
A detailed qualitative examination of the testes was performed on all males in control and high dose groups.
The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment related effects, such as missing germcell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952. The PAS- stained sections were used to identify the spermatogenic stages.

Litter observations:

Parturition and gestation length
A parturition check was performed from Day 20 to Day 25 post coitum.
One female receiving 62.5 mg/kg/day (no. X0740037) did not show any evidence of copulation (mating not detected) during the 14 days of cohabitation with male no. X0740038.
However, this female gave birth and was sacrificed with its litter on Day 14 post partum.
Female no. X0740063 (Group 4), which did not give birth after 25 days of post coitum period, was sacrificed on Day 26 post coitum. This female was found not pregnant at necropsy.
Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth when the parturition was defined complete (Day 0 post partum).

Pups identification, weight and observation
As soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1, 4, 7 and 13 post partum. The data were reported for Days 1, 4 and 13 post partum. The data of Day 7 post partum are not tabulated in this report but will be archived together with all other raw data. Pups killed or dying during the lactation period were weighed before the despatch to necropsy.
Observation was performed once daily for all litters.

Adjustment of litter size (culling) and pups selection for blood collection at necropsy
Culling – Day 4 post partum
On Day 4 post partum, all pups were weighed and litters in excess of 8 offspring were culled to 8 (4 males and 4 females, where possible) by a random selection. At least two pups (males or females, selected for culling) were sacrificed for blood collection. In female no. X0740045 (Group 3), only one male pup was selected.
For litters with 8 pups (nos. X0740049 and X0740035) or less (no. X0740053), two female pups were sacrificed for blood collection.


Anogenital distance (AGD)
The AGD of each pup was measured on Day 1 post partum. The AGD was normalized to the cube root of body weight collected on Day 1 post partum.

Nipple count on Day 13 post partum
No nipples/areolae in male pups were present.

Clinical pathology investigations
4.4.1 Blood collection for thyroid hormone determination (T3, T4 and TSH)
Blood collection from pups on Days 4 and 14 post partum On Day 4 post partum, blood samples (approximately 0.2 mL) were collected from two of the culled pups (males or females, where possible). Blood samples were withdrawn under light ether anaesthesia from the heart (by intracardiac puncture).
On Day 14 post partum, blood samples (approximately 0.5 mL) were withdrawn under light ether anaesthesia from the heart (by intracardiac puncture) from at least two pups (1 sample/sex, if possible) per litter.

Bioanalysis - Thyroid hormone determination (T3, T4 and TSH)
Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by a multiplex assay, using LuminexMagpix system and the MILLIPLEX MAP Rat ThyroidMagnetic Bead Panel kit (MerkMillipore, cat. no. RTHYMAG-30K).
The determination was restricted as detailed below:
1. Samples from all parental males from all groups
2. Samples from pups on Day 14 post partum
The results of these analyses are presented as individual data, mean and standard deviation and reported in ng/mL. Since no treatment related effects were seen in the determination performed in parental males and in pups on Day 14 post partum, samples obtained from females and from pups on Day 4 post partum were not analysed.

Postmortem examinations (parental animals):

Necropsy
The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.
Females
All females were examined also for the following:
– external and internal abnormalities;
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals).
Uteri of apparently non-pregnant female or uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.


Organ weights
Parental animals
From all animals completing the scheduled test period, the organs indicated in Annex were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved
Samples of all the tissues (all parental animals) listed in Annex were fixed and preserved in 10% neutral buffered formalin.

Histopathological examination
The tissues required for histopathological examination are listed in Annex. After dehydration and embedding in paraffin wax, sections of the tissueswere cut at 5 micrometer thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.


The examination was restricted as detailed below:
i Tissues specified in Annex rom all males and females in the control and high dose groups killed at term.
ii Tissues specified in Annex from the animal (no. X074069) dying during the treatment period.
iii All abnormalities in all groups. A detailed qualitative evaluation of testes was performed on all control and high dose males.
The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects, such as: missing germcell layers or types, retained spermatids, multinucleated or apoptotic germcells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and referred to the comprehensive reviews on the subject: Russell, 1990; Creasy, 1997; Creasy, 2002. The PAS- H stained sections were used to identify the spermatogenic stages.

Postmortem examinations (offspring):

Pups
All pups found dead in the cage were examined for external and internal abnormalities.
All culled pups sacrificed on Day 4 post partum were subjected to an external examination.
Sex was determined by internal gonads inspection.
All live pups sacrificed on Day 14 post partum were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
4.5.3 Nipple count retention on Day 14 post partum
The ventral region of male pups was checked for presence of nipples/areolae.
No nipples were found in pups to be retained on Day 14 post partum. Data were not tabulated, but will be archived with all raw data.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopatholgical findings was carried out by means of the non-parametric Kolmogorov Smirnov test.
The non-parametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of theWilliams test. The criterion for statistical significance was p < 0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Reproductive indices:

Presentation of data
Group mean values, where possible, were calculated for all parameters. Data from non pregnant female were excluded from group mean calculations as considered appropriateby the Study Director.
The following reproductive indices were calculated:

Males
Copulation Index (%) = no. of males mated/no. of mal es paired×100
Fertility Index (%) = no. of males which induced pregnancy/no. of males paired×100

Females
Copulatory Index (%) = no. of females mate d/no. of females paired×100
Fertility Index (%) = no. of pregnant females/no.of females paired×100
Males and females
Coital Interval = Mean number of days between pairing and mating

Offspring viability indices:

Females
Pre-implantation loss was calculated as a percentage from the formula: no. of corpora lutea−no. of impl ant a t i ons/no. of corpora lutea×100

Pre-natal loss on Day 0 post partum, before culling, was calculated as a percentage from the formula: no. of visible implantations −live litter size at birt h/no. ofvisible implantations×100

Pup loss at Day 0 post partum was calculated as a percentage from the formula:
Total litter size at birth −live litter size at birth/Total litter size at birthx100


Post-natal loss on Day 4 post partum, before culling, was calculated as a percentage from the formula:
Live litter size at birth −live litter size at Day 4, before culling/Live litter size at birth×100

Post-natal loss on Day 13 post partum (after culling) was calculated as a percentage from the formula: Live litter size on Day 4(after culling)−live litter size on Day 13/Live litter size on Day 4(after culling)×100

Sex ratios were calculated at birth, on Days 4 and 14 post partum and were presented as the percentage of males per litter.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At the daily clinical examination, the following observations were recorded:
– Treated males receiving 250mg/kg/day showed salivation during the study. The sign appeared in all males starting from Day 6 of treatment. One male receiving 125 mg/kg/day (Group 3) showed salivation only on Day 6 of the study.
– Females receiving 250mg/kg/day showed salivation for most of the treatment period. This sign firstly appeared on Day 6 of treatment.

In addition, two females receiving 62.5 mg/kg/day showed scabs on eye(s) and hairloss on lumbar region, respectively. Another female receiving 125 mg/kg/day showed scabs and hairloss on thoracic region.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mortality
One female (no. X0740069) receiving 250 mg/kg/day was found dead on Day 15 of the premating phase (after a total of 14 days of treatment). The clinical signs noted in this animal the day before death were: decreased activity, piloerection, dyspnoea and salivation.
Macroscopically, the findings observed in this animal included: small thymus and incomplete collapse of lungs. Microscopically, the most relevant findingswere interstitial, alveolar, bronchial and perivascular granulomatous reaction associated with oedema and haemorrhage in the lungs. On the basis of these findings, the cause of death of this animal was attributed to a misdosing.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant differences in body weight and body weight gain were noted between control and treated males and females throughout the study.
In particular, on Day 14 of the mating phase, a statistically significant decrease was noted in body weight gain in treated males receiving 250 mg/kg/day when compared to the control group. This change was due to one male (no. X0740080) which showed a decrease in body weight between Days 7 and 14 of the mating phase.

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormones

Parental males
One male dosed at 62.5 mg/kg/day and one receiving 250 mg/kg/day (nos. X0740040 and X0740080, respectively) showed a relevant increase of tyroxine (approximately 4.5 fold compared with controls). Due to the minimal incidence and the absence of other related changes, this finding was considered of no toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Final sacrifice
No treatment-related changes were noted.
The sporadic lesions reported in control and/or treated animals such as spermgranuloma in one epididymis or congestion of thymus in one high dose male and female respectivelywere considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Fate of females
One female receiving 250 mg/kg/day was found not pregnant. Two females, one in the control group and one receiving 62.5 mg/kg/day, showed unilateral implantation in the left horn and were not pregnant in the right one.
In addition, one female receiving 250 mg/kg/day did not have positive identification of mating during the 14 days of cohabitation with the male. However, this female gave birth and was sacrificed with its litter on Day 14 post partum.
The number of females with live pups on Day 14 post partum was: 10 in the control, 10 in the low dose (62.5 mg/kg/day), 10 in the mid-dose (125 mg/kg/day) and 8 in the high dose group (250 mg/kg/day).

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

reproductive function (oestrous cycle)

reproductive performance

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

reproductive function (oestrous cycle)

reproductive performance

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Cold to touch, small and pale appearance were the main clinical signs noted in control and/or treated pups. In addition, found dead and/or missing pups were also observed both in control and treated groups, with similar incidence. One pup at the high dose level (250 mg/kg/day) showed wound on muzzle.

Anogenital distance
No relevant differences were seen between the control and treated pups in anogenital distance.

Mortality / viability:

no mortality observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormones

Pups – Day 14 post partum
Thyroid stimulating hormone was increased in a number of pups of both sexes at the high dose level. Compared with controls, the increases were approximately 2.5 fold. Since no changes of the other hormones were recorded, the above finding was considered to have limited toxicological relevance.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Pups thyroid weight on Day 14 post partum
No differences were noted in thyroid weight between control and treated pups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy findings in decedent pups, in pups sacrificed on Days 4 and 14 post partum and nipple count

Autolysed thoracic organs were observed in one pup of Group 4 (250 mg/kg/day) which died during the lactation period.
No significant signs were recorded in pups sacrificed on Days 4 or 14 post partum.
No nipples were observed in male pups on Day 14 post partum.

Other effects:

no effects observed

Description (incidence and severity):

Implantation sites, pre-implantation loss data, pre-natal loss data and gestation length of females

Gestation periods were similar between treated females and control group. All pregnant dams gave birth on Day 22 post coitum (mean value).
Number of corpora lutea and implantation sites of treated females were comparable to the control group. Pre-implantation loss and pre-natal loss (both expressed as percentage) were slightly higher than the control values. All these changes were not significant, at statistical analysis, and without dose relationship.

Litter data at birth, on Days 1 and 4 post partum (before culling) and on Day 13 post partum (after culling) and sex ratio of pups

Litter data of treated females recorded at birth, on Days 4 and 13 post partum, were comparable to the control group.
Sex ratios at birth, on Days 4 and 14 post partum, did not show differences between groups, when calculated as the percentage of males.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: general toxicity

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: general toxicity

Key result

Reproductive effects observed:

no

Conclusions:

Summary

1.2 Mortality and fate of females

One female (no. X0740069) receiving 250 mg/kg/day was found dead on Day 15 of the premating phase (after a total of 14 days of treatment). Decreased activity, piloerection, dyspnoea and salivation were the clinical signs recorded the day before death.
On the basis of the macroscopic (small thymus and incomplete collapse of lungs) and microscopic examinations (alveolar, bronchial and perivascular granulomatous reaction associated with oedema and haemorrhage in the lungs), the cause of death of this animal was attributed to a misdosing.
One female receiving 250 mg/kg/day was found not pregnant. Two females, one in the control group and one receiving 62.5 mg/kg/day, showed unilateral implantation in the left horn and were not pregnant in the right one.
In addition, one female receiving 250 mg/kg/day did not have positive identification of mating during the 14 days of cohabitation with the male. However, this female gave birth and was sacrificed with its litter on Day 14 post partum.
The number of females with live pups on Day 14 post partum was: 10 in the control, 10 in the low dose (62.5mg/kg/day), 10 in the mid-dose (125mg/kg/day) and 8 in the high dose group (250mg/kg/day).

1.3 Clinical signs

The main clinical sign noted in all treated animals of both sexes receiving 250 mg/kg/day was salivation. This sign appeared in the animals starting from Day 6 of the study. Only one male receiving 125 mg/kg/day showed salivation on Day 6 of the study.

1.4 Body weight and body weight gain

No relevant differences in body weight and body weight gain were noted between control and treated males and females throughout the study.

1.5 Food consumption
No effects on food consumption were observed in males or females.

1.6 Thyroid hormones

Parental males
Two treated males receiving 62.5 and 250 mg/kg/day, respectively showed a relevant increase of tyroxine. However, in the absence of other related changes, this finding was considered of no toxicological significance.


Pups – Day 14 post partum
On Day 14 post partum, thyroid stimulating hormone was increased in a number of pups of both sexes at dose level of 250 mg/kg/day. Since no changes of the other hormones were
recorded, the above finding was considered to have limited toxicological relevance.

1.7 Oestrous cycle, reproductive parameters, pairing combination and mating performance

Oestrous cycle, pre-coital intervals, copulatory index and fertility index did not show intergroup differences.

1.8 Implantation sites, pre-implantation loss data, pre-natal loss data and gestation length of females

No significant differences were observed for these parameters between treated and control groups. All pregnant dams gave birth on Day 22 post coitum (mean value).

1.9 Litter data at birth, on Days 1 and 4 post partum (before culling) and on Day 13 post partum (after culling) and sex ratio of pups

Litter data at birth, on Days 4 and 13 post partum recorded on treated groups were comparable to those of the control group. Sex ratios at birth, on Days 4 and 14 post partum did not show differences between groups, when calculated as the percentage of males.

1.10 Clinical signs of pups

Pre-weaning clinical signs were comparable between treated and control groups.

1.11 Anogenital distance
No relevant differences were seen between the control and treated pups in anogenital distance.

1.12 Necropsy findings in decedent pups, in pups sacrificed on Days 4 and 14 post partum and nipple count

Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect.
No nipples were observed in male pups on Day 14 post partum.

1.13 Pups thyroid weight on Day 14 post partum

No differences were noted in thyroid weight between control and treated pups.

1.14 Terminal body weight and organ weights

Terminal bodyweight did not showrelevant differences between control and treated groups.
Changes noted in the relative organ weights (testes and epididymides) were of low magnitude and no adverse histological findings were observed at microscopic examination, therefore they were considered of no toxicological relevance.

1.15 Macroscopic observations

Final sacrifice
Animals that completed the treatment period and killed at termination did not show relevant macroscopic changes that could be considered treatment-related.

1.16 Microscopic observations

Final sacrifice
No treatment-related changes were noted.

Spermatogenic cycle
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

1.17 Conclusion

On the basis of the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) for general toxicity, reproductive and developmental toxicity was considered to be 250 mg/kg/day, in terms of 50.7% solid content (composition of the test item was about 50% of active ingredient + 50% of water) for males and females.

Executive summary:

The reproductive toxicity of the substance was investigated in a study according to OECD Guideline 421.

The purpose of this study was to provide limited information on toxic effects on Wistar rats of both sexes after repeated dosing with the substance, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring.

All doses (62.5, 125 and 250 mg/kg/day) were administered orally by gavage. The control group received softened water (by reverse osmosis). The dose volume was 5mL/kg body weight.

Males: were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 32 or 33 days.

Females: were treated for 2 weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum (for at least 51 days). One not pregnant female was dosed up to the day before necropsy.

Salivation was the main clinical sign noted in all treated animals of both sexes receiving 250 mg/kg/day. No differences were noted in the body weights and food consumption between control and treated groups.

Concerning the reproductive parameters, no relevant differences were found in terms of mating performance including the pre-coital interval (number of days paired to sperm positive day), copulatory evidence (the positive identification of mating i.e. the presence of sperm and/or copulation plug in situ or in the cage) or fertility index. All pregnant females had a comparable length of gestation and gave birth on Day 22 post coitum (mean value). Litter data and sex ratios were unaffected by treatment. Similar clinical signs were recorded in control and treated pups during the lactation period. No relevant differences were seen between the control and treated pups in anogenital distance. Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect, as well as in thyroid weight of pups sacrificed on Day 14 post partum.

Two treated parental males receiving 62.5 and 250 mg/kg/day showed a relevant increase of tyroxine. However, in the absence of other related changes, this finding was considered of no toxicological significance. Thyroid stimulating hormone was increased in a number of pups of both sexes at dose level of 250 mg/kg/day. Since no changes of the other hormones were recorded, the above finding was considered to have limited toxicological relevance.

Terminal bodyweight did not showrelevant differences between control and treated groups.

Changes noted in testes and epididymides weights (relative) were of low magnitude and no adverse histological findings were observed at microscopic examination, therefore they were considered of no toxicological relevance.

No treatment-related changes were noted at macroscopic and microscopic observations, as well as on the evaluation of the seminiferous tubules respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages.

On the basis of the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) for general toxicity, reproductive and developmental toxicity was considered to be 250 mg/kg/day (based on active ingredient.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was conducted according to OECD guideline and GLP compliant and is thus of high quality

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 reproductive toxicity of the substance was investigated in a study according to OECD Guideline 421.

The purpose of this study was to provide limited information on toxic effects on Wistar rats of both sexes after repeated dosing with the substance, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring.

All doses (62.5, 125 and 250 mg/kg/day) were administered orally by gavage. The control group received softened water (by reverse osmosis). The dose volume was 5mL/kg body weight.

Males: were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 32 or 33 days.

Females: were treated for 2 weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum (for at least 51 days). One not pregnant female was dosed up to the day before necropsy.

Salivation was the main clinical sign noted in all treated animals of both sexes receiving 250 mg/kg/day. No differences were noted in the body weights and food consumption between control and treated groups.

Concerning the reproductive parameters, no relevant differences were found in terms of mating performance including the pre-coital interval (number of days paired to sperm positive day), copulatory evidence (the positive identification of mating i.e. the presence of sperm and/or copulation plug in situ or in the cage) or fertility index. All pregnant females had a comparable length of gestation and gave birth on Day 22 post coitum (mean value). Litter data and sex ratios were unaffected by treatment. Similar clinical signs were recorded in control and treated pups during the lactation period. No relevant differences were seen between the control and treated pups in anogenital distance. Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect, as well as in thyroid weight of pups sacrificed on Day 14 post partum.

Two treated parental males receiving 62.5 and 250 mg/kg/day showed a relevant increase of tyroxine. However, in the absence of other related changes, this finding was considered of no toxicological significance. Thyroid stimulating hormone was increased in a number of pups of both sexes at dose level of 250 mg/kg/day. Since no changes of the other hormones were recorded, the above finding was considered to have limited toxicological relevance.

Terminal bodyweight did not showrelevant differences between control and treated groups.

Changes noted in testes and epididymides weights (relative) were of low magnitude and no adverse histological findings were observed at microscopic examination, therefore they were considered of no toxicological relevance.

No treatment-related changes were noted at macroscopic and microscopic observations, as well as on the evaluation of the seminiferous tubules respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages.

On the basis of the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) for general toxicity, reproductive and developmental toxicity was considered to be 250 mg/kg/day (based on test material), i.e. 125 mg/kg bw/d (based on active ingredient).

Effects on developmental toxicity

Description of key information

-study conducted according to OECD guideline 421 (adopted 29 July 2016), male and female Wistar rats were exposed to 0, 62.5, 125, 250 mg/kg bw /d aqueous solution of Cocobetainamidoamphopropionate (a.i. 50%) by oral gavage for 32 or 33 days and at least 51 days, respectively, no treatment related effects were observed up to the highest dose tested, NOAEL = 250 mg/kg bw/day (a.i. 50%)

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was conducted according to OECD guideline and GLP compliant and is thus of high quality

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 reproductive toxicity of the substance was investigated in a study according to OECD Guideline 421.

The purpose of this study was to provide limited information on toxic effects on Wistar rats of both sexes after repeated dosing with the substance, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring.

All doses (62.5, 125 and 250 mg/kg/day) were administered orally by gavage. The control group received softened water (by reverse osmosis). The dose volume was 5mL/kg body weight.

Males: were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 32 or 33 days.

Females: were treated for 2 weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum (for at least 51 days). One not pregnant female was dosed up to the day before necropsy.

Salivation was the main clinical sign noted in all treated animals of both sexes receiving 250 mg/kg/day. No differences were noted in the body weights and food consumption between control and treated groups.

Concerning the reproductive parameters, no relevant differences were found in terms of mating performance including the pre-coital interval (number of days paired to sperm positive day), copulatory evidence (the positive identification of mating i.e. the presence of sperm and/or copulation plug in situ or in the cage) or fertility index. All pregnant females had a comparable length of gestation and gave birth on Day 22 post coitum (mean value). Litter data and sex ratios were unaffected by treatment. Similar clinical signs were recorded in control and treated pups during the lactation period. No relevant differences were seen between the control and treated pups in anogenital distance. Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect, as well as in thyroid weight of pups sacrificed on Day 14 post partum.

Two treated parental males receiving 62.5 and 250 mg/kg/day showed a relevant increase of tyroxine. However, in the absence of other related changes, this finding was considered of no toxicological significance. Thyroid stimulating hormone was increased in a number of pups of both sexes at dose level of 250 mg/kg/day. Since no changes of the other hormones were recorded, the above finding was considered to have limited toxicological relevance.

Terminal bodyweight did not showrelevant differences between control and treated groups.

Changes noted in testes and epididymides weights (relative) were of low magnitude and no adverse histological findings were observed at microscopic examination, therefore they were considered of no toxicological relevance.

No treatment-related changes were noted at macroscopic and microscopic observations, as well as on the evaluation of the seminiferous tubules respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages.

On the basis of the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) for general toxicity, reproductive and developmental toxicity was considered to be 250 mg/kg/day (based on test material), i.e. 125 mg/kg bw/d (based on active ingredient).

Justification for classification or non-classification

Based on the the relevant and available data the test item does not need to be classified according to Regulation (EC) No 1272/2008 (CLP and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS) with respect to reproductive/developmental toxicity.