Fatty acids, C18 unsatd., mono- and diesters with triethanolamine, di-Me sulfate quaternized

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 January 2020 - 08 March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

Fatty acids, C16-18(even numbered) and C18 unsatd., reaction products with triethanolamine, di-Me sulfate-quaternized

Cas Number:

91995-81-2 / 157905-74-3 / 93334-15-7

Molecular formula:

n.a. (UVCB)

IUPAC Name:

Fatty acids, C16-18(even numbered) and C18 unsatd., reaction products with triethanolamine, di-Me sulfate-quaternized

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age and weight at study initiation: 7 to 8 weeks old and
weighing 200 to 225 g for males and 175 to 200 g for females
- Fasting period before study: no
- Housing:
from arrival to mating: up to 5 of one sex to a cage, in polysulfone
solid bottomed cages measuring 59.5×38×20cm (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: one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5cm with a stainless steel mesh lid and floor (Tecniplast Gazzada
S.a.r.l., Buguggiate, Varese)
After mating: males were re-caged as they were before mating; 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 0Mucedola) was provided as necessary. Nesting material was changed at least two times a week.
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 32 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±15
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
-dosing solutions were prepared in concentrations of 10, 30 and 100 mg/mL

- dose volume: 10mL/kg body weight

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until positive identification occurred
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Results of the analyses were within the acceptability limits for suspensions
(85-115% for concentration and CV < 10% for homogeneity)

Duration of treatment / exposure:

males: for 14 days prior to pairing and thereafter until the day before necropsy, for a total of 30 days
females: 14 days prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum, for a total of 50 to 55 days

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated (screenign study)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

MORTALITY: Yes
twice daily

CLINICAL SIGNS: Yes
Once before commencement of treatment and at least once daily during the study


BODY WEIGHT: Yes
- Time schedule for examinations:
Males: weekly from allocation to termination
Females: weekly from allocation to positive identification of mating and on
gestation Days 0, 7, 14 and 20; Dams: Days 1, 4, 7 and 13 post partum and on the day of necropsy


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


WATER CONSUMPTION: No

OTHER:
thyroid hormone determination (T3, T4 and TSH)
Males at termination
Females at termination
Pups on Days 4 and 13 or 14 post partum

Blood collection for detection of test item - Dams and pups
Dams
Blood samples (approximately 0.3 mL) were taken from the tail vein of 5 females/group on Day 18 post coitum and Day 13 post partum at 1 time point (4 hours post-dose).
Pups
On Day 13 post partum blood samples (approximately 0.3 mL) were taken from 1 pup/sex/litter from 5 selected dams of each group (5 pups/sex/group). Blood samples were withdrawn at necropsy under light ether anaesthesia from the heart (intracardiac puncture).

Oestrous cyclicity (parental animals):

Before allocation (stock females)
Oestrous cycle was monitored by vaginal smears in all females ordered for the study for 2 weeks before allocation, in order to exclude females showing irregular oestrus cyclicity.

After allocation (females allocated to groups)
Vaginal smears were taken in the morning from Day 1 of dosing up to positive identification of copulation. The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle;
2. pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

Before despatch to necropsy
Vaginal smears were also taken from all females before despatch to necropsy and the oestrous cycle phase recorded.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- size of each litter was adjusted by eliminating extra pups by random
selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment was acceptable. At least one culled male and one
culled female was selected for hormone determination with the exception of the following females where the selection was performed on the basis of the pups available:
- Group 1 : female no. X12500005: 2 female pups selected
female no. X12500009: 1 male pups selected
- Group 3 : female no. X12500041: 1 female pup selected
female no. X12500047: 1 female pup selected
- Group 4 : female no. X12500077: 2 female pups selected

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

thyroid hormone determination (T3, T4 and TSH): Pups on Days 4 and 13 or 14 post partum

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals 30 days of treatment.
- Maternal animals: All surviving animals on Day 14 post partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera]

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examinations

– Internal examination - Gonads were inspected from all pups in order to confirmthe
sex previously determined by external examination.
– Nipples retention at Day 14 post partum - 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.

HISTOPATHOLOGY / ORGAN WEIGTHS
thyroid weights

Statistics:

Standard deviationswere calculated as appropriate. For variables such as bodyweight, food consumption, hormone determination and organ weight 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 histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
Intergroup differences between the control and treated groups were assessed by the nonparametric version of theWilliams test. The criterion for statistical significance was p<0.05.

Reproductive indices:

Copulation Index
Fertility Index
Pre-implantation loss
Pre-natal loss

Offspring viability indices:

Post-natal loss at Day 0 post partum
Post-natal loss at Day 4 post partum (before culling)
Post natal loss at Day 13 post partum (after culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was the treatment-related clinical sign recorded during the study. This sign was occasionally noticed in all males and females treated at 1000mg/kg/day, starting from the pre-mating phase until sacrifice (end of mating for males and post partum phase for females). Males treated at 300mg/kg/day also showed salivation, but this sign appeared later in the study, mostly at the end of the mating was considered not adverse.
Other signs, such as scabs, damaged eye, hairloss and isolation in cage, were sporadically recorded during the study. These findings were considered as incidental or related to in-life procedures and were, thus, deemed as minor clinical signs, not related to treatment with the test item.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No differences in body weight were observed in treated animals of both sexes, when compared to controls.
Statistically significant differences in body weight gain were observed:
– decrease in low and high dose groups of males on Day 8 of treatment
– decrease in low dose group of females on Day 1 of treatment
– increase in high dose females on Day 7 post partum
These differences were occasional and with no dose relationship, therefore considered unrelated to treatment.

Food consumption and compound intake (if feeding study):

no effects observed

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

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid hormones: No changes were recorded between control and treated groups.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were noted during the microscopic organ exminations.
The sporadic lesions, reported in control and treated animals, were considered to be an expression of spontaneous/or incidental pathology, seen in this species and age in this kind of studies and under the test site's experimental conditions.

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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

Fate of females
All females mated.
The number of females with live pups on Day 13/14 post partum was 10 in the control group and 10 in all treated groups, dosed at 100, 300 and 1000 mg/kg/day.

Oestrous cycle, reproductive parameters, pairing combination and mating
performance
No anomalies were noted in the oestrous cycle and pre-coital interval of the treated females, when compared to controls.
All females mated, all gave birth and all conceivings were within 5 days of mating.

The copulatory indices were 100% for controls and treated animals. The fertility indices were 100% for control animals and 100% for all treated groups.

Implantation sites, pre-implantation loss data, pre-natal loss dataandgestation
length of females
Implantations and pre-implantation loss, total litter size and pre-natal loss (as percentage) did not show dose-related or treatment-related differences.
Gestation periods were comparable between control and treated groups. All dams gave birth between Days 21 and 23 post coitum.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no signs of treatment-related toxicity

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Small size, cold to touch, pallor, apparently no food intake (milk) and small appearance were in general the clinical signs noted in the pups of control and treated groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No significant differences in total litter size, live litter size, mean pup loss (percentage), and sex ratio of pups were observed among the treated and the control dams at birth and on Days 1, 4, 13 and 14 post partum.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant differences in mean pup weights (percentage) were observed among the treated and the control dams at birth and on Days 1, 4, 13 and 14 post partum.

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

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant decreases in the anogenital distances were noted in male pups (up to 14%) and female pups (up to 32%) of treated groups in respect to the controls. However, the above results were considered incidental and unrelated to treatment, considering the following:

- all values were within the range of ERBC historical control data (0.55 - 3.14mm/g1⁄3 for males and 0.48 - 2.09mm/g1⁄3 for females). What are the control group means of the historical data? Compare mean litter values with mean litter values of historical control data. To might be able to show that It Is some individual litter causing the decrease, and not a decrease in all litters.
- there was no relation to the dose. (Compound identified as endocrine disruptors show marked effects on AGD in a dose-dependent manner. Camilla Lindgren Schwartz, et al., Archives of Toxicology volume 93, pages253–272(2019)) – Absence of other correlated findings considered androgen-mediated endpoints (such as areola/nipple retention, cryptorchidism, decreased reproductive organ weights, and malformation incidence)
- the testing was performed at high doses, not representing real live exposure ("For these evaluations, there should be stronger emphasis on effect doses and to what extend supra-high test reliably recapitulate what occurs at more human relevant doses." Camilla Lindgren Schwartz, et al., Archives of Toxicology volume 93, pages253–272(2019)).

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were observed in male pups on Day 13/14 post partum.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant differences were noted in thyroid weight between controls and pups of treated groups (1 pup/sex/litter).

Gross pathological findings:

no effects observed

Description (incidence and severity):

The majority of decedent pups from all groups were found autolysed or cannibalised without any possibility for a macroscopic examination of internal organs. However, at external examination, performed in pups not extremely cannibalised, no findings were noted.
Culled pups sacrificed on Day 4 post partum and those sacrificed onDays 13/14 post partum did not show any abnormalities.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormones in Pups - Day 13/14 post partum
No changes were recorded between control and treated groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no signs of treatment-related toxicity

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

REPRODUCTIVE PARAMETERS OF ANIMALS – SUMMARY

 

	Group	1	2	3	4
Copulatory Index% (males)		100.0	100.0	100.0	100.0
Fertility Index% (males)		100.0	100.0	100.0	100.0
Copulatory Index% (females)		100.0	100.0	100.0	100.0
Fertility Index% (females)		100.0	100.0	100.0	100.0

 

 

IMPLANTATION, PRE-IMPLANTATION LOSS DATA, PRE-NATAL LOSS DATA AND GESTATION LENGTH OF FEMALES - GROUP MEAN DATA

 

Group		Corpora Lutea	Implantations	Pre-implantation loss %	Live Litter size at birth	Pre-natal loss %	Gestation length (days)
1	Mean SD (n)	18.20 1.62 10	18.10 1.66 10	0.56 1.77 10	16.80 2.25 10	7.23 8.75 10	22.10 0.32 10
2	Mean SD (n)	16.80 3.19 10	16.80 3.19 10	0.00 0.00 10	15.00 3.02 10	10.11 10.74 10	21.90 0.32 10
3	Mean SD (n)	16.20 2.98 10	16.00 3.53 10	2.22 7.02 10	14.00 4.16 10	13.26 14.25 10	22.10 0.32 10
4	Mean SD (n)	17.00 2.58 10	16.70 2.67 10	1.82 2.94 10	15.50 2.51 10	7.08 8.55 10	22.10 0.32 10

Statistical analysis: Kruskall Wallis test

William’s test if group mean differences are different from control at p < 0.05

* = mean value of group is significantly different from control

Applicant's summary and conclusion

Conclusions:

The toxic effects on Sprague Dawley rats of both sexes were investigated after repeated dosing with the test item. Furthermore, effects of the test item on male and female reproductive performance were examined, i.e. gonadal function, mating behaviour, conception, development of conceptus, parturition and early lactation of the offspring.
Groups of 10 males and 10 females received the test item, by gavage, at dosages of 0, 100, 300 and 1000 mg/kg body weight/day. An additional group of 10 males and 10 females received the vehicle alone (softened water) at the dose volume of 10 mL/kg and acted as control.
No effects were observed in the in vivo parameters of parental animals: no toxicologically relevant clinical signs and no effects on body weight, body weight gain and food consumption were noted at any dose level investigated. Thyroid hormones (T3, T4 and TSH) evaluated in parental males did not show any changes of toxicological relevance. No remarkable differences were noted at post mortem examination including organ weights and no treatment-related macroscopic and microscopic changes were observed in treated animals, when compared to controls. Futhermore, no effects on the spermatogenic cycle were described. No intergroup differences were seen in oestrous cycle, pre-coital intervals, copulatory and fertility indices.
No significant differences were observed in the number of corpora lutea, implantations, total litter size, pre-implantation loss, pre-birth loss and gestation length between control and treated females. Litter data at birth and on Days 1, 4 and 13 post partum and sex ratios were also comparable between treated and control females. There were neither treatment-related signs at clinical observation of pups, nor at necropsy of deceased pups or those sacrificed after culling or at term. The statistically significant decrease noted in the ano-genital distance of male and female pups on Day 1 of age in all treated groups was not considered of toxicological relevance, as there was no clear relation to the dose and as all values were within ERBC historical control data. No nipples were observed on Day 13 of age, in male pups at all dose levels. Thyroid hormones levels and pups thyroid weight were unaffected by treatment.
Analysis of plasma samples collected at 4 hours post-dose, showed that after oral administration of PC-2019-848, parental females were systemically exposed to the test item (detected as the physiological degradation product TEA-Core) both on Day 18 post coitum and Day 13 post partum. The systemic exposure increased with the dose but not with the duration of treatment. Systemic exposure was also considered demonstrated in not dosed pups on Day 13 post partum, although with values below the quantification limits (BLOQ).
Based on the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) for both general toxicity and reproduction/developmental toxicity was considered to be 1000 mg/kg/day for both males and females.

Executive summary:

 The toxic effects on Sprague Dawley rats of both sexes were investigated after repeated dosing with the test item partially unsaturated TEA-Esterquat in accordance with OECD Guideline 421 (adopted 29  July 2016).

Furthermore, effects of the test item on male and female reproductive performance were examined, i.e. gonadal function, mating behaviour, conception, parturition and early lactation of the offspring.

The vehicle was water (was water softened by reverse osmosis. All doses (0, 100, 300 and 1000 mg/kg/day) were administered orally, by gavage at a dose volume of 10mL/kg body weight.

 

Males were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, for a total of 30 days. Females were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum.

The following investigations were performed: body weight, body weight gain, clinical signs, food consumption, macroscopic observations, organ weights and histopathological examination.

In addition he following assessments performed: oestrous cycle evaluation for parental females (2 weeks before start of dosing, during pre-mating and mating phases, prior to necropsy), mating performance, thyroid hormone determination (parental males and pups at Day 13/14 post partum) and collection of litter data.

Clinical signs, anogenital distance, external and/or internal examination were recorded for pups. Thyroid hormone levels and thyroid weight were also determined in 1 pup/sex/group randomly selected at Day 14 post partum.

Routine histopathological examination was performed only in control and high dose groups and it included identification of the stages of the spermatogenic cycle in male animals.

 

1.2 Fate of females

Details of the pregnancy status were as follows:

Groups                                                                         1          2          3          4

Non-pregnant females                                                   0          0          0          0

Conceiving 1 - 5 days                                                    10        10        10        10

No. of females with live pups on Day 13/14 post partum 10         10        10        10

 

1.3 Mortality and clinical signs

No mortality occurred during the study.

Salivation was the most relevant and treatment-related clinical sign recorded during the

study. This sign was occasionally noticed in all males and females treated at 1000mg/kg/day,

starting from the pre-mating phase until sacrifice (end of mating for males and post partum

phase for females). Males treated at 300mg/kg/day also showed salivation, but this sign

appeared later in the study, mostly at the end of the mating phase.

 

1.4 Body weight and body weight gain

No differences in body weight were observed in treated animals of both sexes, when compared

to controls and no differences considered treatment-related were observed in body

weight gain of treated animals.

 

1.5 Food consumption

Food consumption was unaffected by treatment.

 

1.6 Blood detection of test item

Systemic exposure of pups through the mother milk was also demonstrated by the presence of the physiological degradation product TEA-Core in pups on Day 13 post partum, i.e. prior to weaning.  However, quantification was not possible since the detected values of TEA-Core were below the limit of quantification (BLOQ).

 

1.7 Oestrous cycle, reproductive parameters, pairing combination and mating

performance

Oestrous cycles, pre-coital interval, copulatory index and fertility index did not show any

treatment-related intergroup differences.

 

1.8 Implantation sites, pre-implantation loss data, pre-natal loss data And gestation

length of females

Implantation sites, pre-implantation and pre-natal loss and gestation length did not show treatment-related differences.

 

1.9 Litter data and sex ratio of pups

No significant differences in total and live litter size, pup loss, litter weights and mean pup weight were observed among treated and control females at birth and on Days 1, 4 and 13 post partum. Sex ratio did not show any significant differences between groups.

 

1.10 Clinical signs of pups

No signs considered treatment related were seen in pups of treated groups.

 

1.11 Anogenital distance

The statistically significant decrease noted in the ano-genital distance of male and female pups on Day 1 of age in all treated groups was considered incidental and unrelated to treatment, considering that: in both sexes all values were within the range of ERBC historical control data and there was no relation to the dose.

In addition, in females, the decrease observed represents a trend towards feminisation and is not considered an adverse effect (i.e. an increase in AGD of females, suggesting masculinisation); in males, there was no correlations with other findings considered androgen-mediated endpoints (such as areola/nipple retention, cryptorchidism, decreased reproductive organ weights, and malformation incidence).

 

1.12 Thyroid hormones

Parental animals

No differences were recorded between treated and control groups.

Pups - Day 14 post partum

No differences were recorded between treated and control groups.

 

1.13 Necropsy findings in decedent pups, inpupssacrificedonDays 4and13/14

post partum and nipple count

No findings were seen at necropsy in decedent pups or in those sacrificed on Days 4 and 13 post partum.

No nipples were observed in male pups on Day 13 post partum.

 

1.14 Pups thyroid weight on Day 13/14 post partum

No significant differences were noted in thyroid weight between controls and pups of treated groups.

 

1.15 Terminal body weight and organ weights

No changes attributable to the treatment were observed in terminal body, absolute and relative organ weight of treated animals, when compared to the controls.

 

1.16 Macroscopic observations

No significant differences were noted at post mortem examination in treated animals, when compared to the controls.

 

1.17 Microscopic observations

No treatment-related changes were observed in treated animals, when compared to the controls.

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.

 

1.18 Conclusion

In conclusion, no signs of treatment-related toxicity were observed at any of the dose levels investigated following treatment with the test item, when administered to rats by oral route at dose levels of 100, 300 and 1000 mg/kg/day. Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general, reproductive and developmental toxicity was considered to be 1000 mg/kg/day for males and females.