7-anilino-4-hydroxy-3-[[6-methoxy-4-[(6-sulpho-2,4-xylyl)azo]-m-tolyl]azo]naphthalene-2-sulphonic acid, sodium salt, compound with 2,2',2''-nitrilotriethanol

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

repeated dose reproduction study OECD 422

Type of information:

experimental study

Remarks:

source of read across

Adequacy of study:

key study

Study period:

12 April 2017 to

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Specific details on test material used for the study:

grounded to powder prior to formluation

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD) rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: Males: ca 72 days; Females: ca 86 days
- Weight at study initiation: Males: 345 to 415 g; Females: 236 to 298 g.
- Fasting period before study: no
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid and a solid bottom (during pairing grid bottomed polypropylene cages)
Pre-pairing up to five animals of one sex
Pairing one male and one female
Males after mating up to five animals
Gestation one female
Lactation one female + litter
- Enrichment: Aspen chew block and Plastic shelter (except during pairing and lactation)
- Diet: SDS VRF1 Certified pelleted diet ad libitum
- Water: potable public water ad libitum
- Acclimation period: males 6 days; females 20 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24ºC:
- Humidity: 40-70 %
- Air changes (per hr): no data: filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

using a suitably graduated syringe and a rubber catheter inserted via the mouth (dosing volume adjusted to latest measured body weight)

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was ground before weighing out the required amount. Approximately 50% of the required volume of vehicle was added to the test item and it was magnetically stirred for a minimum of one hour to ensure accurate mixing. The remaining amount of vehicle was added and it was magnetically stirred for a minimum of 20 minutes.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

VEHICLE:water
- Concentration in vehicle: 0, 1, 3 and 8 mg/mL
- Amount of vehicle (if gavage): 5 mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the formulations (1 mL, accurately weighed) were dissolved using ultrasonic vibration in a suitable volume of water and refrigirated for 1 day to allow full dissolution of substance before re-equilibration to ambient temperature prior to dilution. Once at room temperature the extract was diluted to provide a solution containing the substance at an expected concentration within the range 2 µg/mL to 4 µg/mL. These solutions were anaylsed by HPLC/UV.

HPLC: Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: Agilent Poroshell C18-SB, 2.7 µm, 100 × 4.6 mm
Column temperature: 45ºC
Sample temperature: Ambient
Mobile Phase A: ACN/10mM ammonium formate 10/90 v/v
Mobile Phase B: ACN/10mM ammonium formate 90/10 v/v
Gradient: Time (min) A(%) B(%)
0.0 95 5
0.5 95 5
5.0 20 80
5.2 20 80
5.3 95 5
8.0 95 5

Flow rate: 1.0 mL/min
Detector wavelength: UV, 560 nm
Injection volume: 10 µL
Run time: 8 minutes
Approximate retention time:3.7 minutes

Calibration (1.0 to 5.01 ug/mL): y= 41812x + 257 (R2=0.99980) (standards 1 and 5 ug/ml CV% 0.86 and 0,53)
Procedural recovery (validation) at 1 and 100 mg/mL: 100.5-102.5% of nominal
Procedural recovery (test solutions) at 1 and 100 mg/L on day 0,1 and 15: 94.9-104.4% of nominal; on day 8 90-100.9% of nominal
Stability at 21˚C over 15 days; at 1 and 100 mg/L: 108 and 114% of nominal
Stability (refrigerated) over 8 dys: at 100 mg/L congealed on Day 8
Homogeneity at 1 and 100 mg/L: CV% <0.87 and <1.44
Analyzed Concentrations: 85.5- 100.5 % of nominal (see attached table)

Duration of treatment / exposure:

Males Two weeks pre-pairing up to necropsy after a minimum of five weeks of treatment (animals were killed in Week 6).
Females Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

40 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 2-week dose range finding study
In that study, treatment at 100, 250 or 500 mg/kg/day was not tolerated and resulted in one or more animals either found dead without any prior warning of a decline in clinical condition, or killed for reasons of animal welfare. Treatment at 50 mg/kg/day was tolerated, but body weight losses were recorded for one out of three males and one out of three females (19g and 6g respectively) during the second week of treatment and thus suggests an effect of treatment. Taking into consideration the much larger group size, the much longer duration of treatment and the increased physiological demand on the females during pregnancy and lactation on this main OCED 422 study; the high dose was limited to 40 mg/kg/day as the dose was expected to be tolerated but may elicit signs of toxicity manifesting as initial body weight loss in some animals. The intermediate and low doses were set at 15 and 5 mg/kg/day to permit evaluation of any dose related trends over an approximate geometric progression of doses.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: pre-dose, post-dose and at the end of the day
F0 males Week 1 - daily
Week 2 onwards - once each week
F0 females Week 1 - daily
Week 2 - once
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: in a standard arena before treatment commenced and during each week of treatment and for females on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males Weekly during acclimatization.
Before dosing on the day that treatment commenced (Day 1) and weekly thereafter.
On the day of necrospy.
F0 females Weekly during acclimatization.
Before dosing on the day that treatment commenced (Day 1) and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7 and 13 of lactation.
On the day of necropsy.

FOOD CONSUMPTION : Yes
- Time schedule for examinations:
Weekly, from the day that treatment commenced until animals paired for mating.
For females after mating food consumption was performed to match the body weight recording:
Days 0-6, 7-13 and 14-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.

Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes overnight
- How many animals: 5/sex/group
- Parameters checked: Hematocrit (Hct), Hemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell hemoglobin (MCH), Mean cell hemoglobin concentration (MCHC)*, Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count:, Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes overnight
- How many animals: 5/sex/group
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Bile acids (Bi Ac), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males in week 5, females at day 7-9 of lactation
- Dose groups that were examined: 5/sex/group
- Battery of functions tested: sensory activity (includes: approach response, pinna reflex, auditory startle reflex, tail pinch)/ grip strength (fore- and hindlimb) / motor activity(beam crossing over 6 min intervals for 1 hour)

IMMUNOLOGY: Yes
- Time schedule for examinations: at termination
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes overnight
- How many animals: all adult males
- Dose groups that were examined: all
- Parameters checked: serum samples of the adult males for thyroxine (T4) levels (additional taken samples from all adult females were not further examined)

ESTOUS CYCLE: Yes
Dry smears For 15 days before pairing using cotton swabs.
Wet smears Using pipette lavage during the following phases:
-For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
-After pairing until mating.
-For four days before scheduled termination.

Sacrifice and pathology:

ORGAN WEIGHTS: Yes (see tables)

GROSS PATHOLOGY: Yes (see tables)

HISTOPATHOLOGY: Yes (see tables)
Premature deaths All F0 animals from all groups.
Scheduled kill F0 animals in Groups 1 and 4:
All F0 animals. Abnormalities only.
The five lowest numbered surviving F0 males with a surviving litter in Groups 2 and 3: Testes and epididymides.

Other examinations:

F0 Females:
Each ovary:Number of corpora lutea
Each uterine horn: Number of uterine implantation sites were recorded.

A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted

Statistics:

The following sequence of statistical tests was used for grip strength, motor activity, body weight, food consumption, clinical pathology, organ weight data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level.
For all other comparisons the F1 approximate test was applied (Williams 1971, 1972). If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations.
For all other comparisons the H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. This test is designed to be used when the main test for comparison of the means is a non-parametric monotonic trend test, such as Shirley's test (Shirley 1977).

For grip strength, motor activity, clinical pathology, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.

For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied.
Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

no signs seen in association with the dosing procedure.
females: dark brown discoloration of dorsal surface in controls, 5 and 15 mg/kg bw

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Incidental
1 female at 15 mg/kg bw: on day 14 of gestation (rupture of a mass on the upper ventral surface; this mass was found to correlate with a mammary adenocarcinoma)
1 male at 40 mg/k bw:day 27(dosing error)

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

males: no treatment related effects
females: BW increased during gestation and/or lactation at 5, 15 and 40 mg/kg bw (can be attributed to low control values)
BW gain was decreased at 40 mg/kg bw during gestation and lactation

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment elated effects

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

males: slightly low hematocrit counts attaining statistical significance at 15 or 40 mg/kg/day ( both -9.6%)
females: at 40 mg/kg bw sign increased MCV (+5%) and sign decreasd PT (-20%); increase in WBC with significant increase of lymphocytes (+75%)

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

For females on Day 14 of lactation, data were variable with atypically high results observed in many parameters for the control females, such as Urea (13.05 - 35.53 mmol/L) or Creatinine (Mean: 38 - 112 μmol/L). These findings were consistent with the observed mineralization identified at histopathological examination (See Section 6.3.12) across all study groups, predominantly in control females.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Sensory reactivity observations and grip strength: no treatment related effects
Motor activity: in males receiving 40 mg/kg/day was low when compared with the controls. Statistical significances were attained at the first of the 6-minute intervals and total scores for high beams

see table

Immunological findings:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males (see table). The control value was relatively low.
The microscopic examination of thymus, thyroid, adrenal and pituitary glands was also unremarkable.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

females: 5, 15 and 40 mg/kg bw sign increased ovary weight (14, 12 and 20%, no clear relationship with dose)
Females: 40 mg/kg bw sign increased uterus weight (27%)

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment related effects
Pale areas on kidney and heart correlated with mineralization (especially seen in control animals)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males
Microscopic examination performed after 5 weeks of treatment revealed minimal to slight degeneration/atrophy in the testes in 4 / 9 males treated at 40 mg/kg/day. A minimal increase in luminal cell debris in the epididymides was seen in the majority of males treated at 40 mg/kg/day; this was also present in one animal given 15 mg/kg/day, but at the incidence and severity were within historical control ranges (0-20%). In three animals given 40 mg/kg/day, the luminal cell debris were associated with minimal to moderate interstitial inflammatory cell infiltrate (see table)

Females
Microscopic examination performed on Day 14 of lactation revealed lesions in the heart (myocardial/vascular mineralization), kidneys (cortical tubular degeneration/necrosis, mineralization) and stomach (mucosal necrosis, mineralization) in several females, including in control animals with no relationship to treatment. The microscopic findings in the heart of myocardial/vascular mineralisation correlated with the pale areas seen macroscopically. Cortical tubular degeneration/necrosis and mineralization seen in the kidneys correlated with pale areas seen at necropsy. Mucosal necrosis and mineralization were also seen in the glandular stomach. Dark areas seen in the stomach macroscopically correlated with the mucosal erosion/congestion seen microscopically.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

no treatment related effect (oestrus cycle 4-5 days pre-treatment and at termination

Details on results:

The oral administration of Direct Violet 35 to Sprague-Dawley rats at dose levels of 5, 15 or 40 mg/kg/day was generally well tolerated with no treatment related mortalities and no adverse effects of treatment on clinical condition, sensory reactivity, grip strength, body weight gain, food intake, haematology or blood chemistry measurements in males and females.
Minimal increase in luminal cell debris associated with minimal to slight degeneration/ atrophy in the testes was seen in animals given 40 mg/kg/day. The change in the testes was seen mostly in late stage tubules, with minimal decrease in germ cells, with no cell or stage abnormalities in particular being noted. In addition, treatment at 40 mg/kg/day was also associated with a minimal increase in luminal cell debris in the epididymides which was seen in the majority of animals In three animals given 40 mg/kg/day, the luminal cell debris were associated with minimal to moderate interstitial inflammatory cell infiltrate. A slight edema was presented in one animal.
A reduction in fertility at 40 mg/kg/day was also evident with only 5 / 10 females pregnant however there was no correlation between the findings in the testes and those females which were found not pregnant; as such it remains unclear if the fertility was attributed to the males or the females treated with the substance. Apart from the reduction in fertility, there was no systemic toxicity detected in females treated with the substance.

Effect levels

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: absence of adverse effects

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

40 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

cauda epididymis

testes

Treatment related:

yes

Any other information on results incl. tables

Sensory reactivity observations and grip strength

Group/sex:		Statistics	1M	2M	3M	4M	1F	2F	3F	4F
Number of animals:		test (M)	5	5	5	5	5	5	5	4
Parameter	Grade
Approach response (1-3)	2		5	5	5	5	5	5	5	4
Pinna reflex (1-3)	2		5	5	5	5	5	5	5	4
Auditory startle reflex (1-4)	2		1	1	1	0
	3		4	4	4	5	4	5	5	4
	4						1	0	0	0
Tail pinch response (1-4)	2		0	0	1	0
	3		5	5	4	5	4	4	5	4
	4						1	1	0	0
Forelimb grip strength (kg)	Mean	Wi	1.05	1.02	1.13	1.02	0.98	1.06	1.09	1.05
	SD		0.03	0.05	0.10	0.06	0.08	0.09	0.07	0.04
Hindlimb grip strength (kg)	Mean	Wi	0.47	0.45	0.50	0.45	0.46	0.46	0.51	0.45
	SD		0.02	0.03	0.05	0.04	0.05	0.04	0.03	0.02

 

Motor activity - group mean scores (beam breaks) (F0)  

Group /Sex	Number of animals	Beam level		Group /Sex	Number of animals
			Total			Total
Statistics test Wiliams			Wi
1M	5	High	194.8	1F	5	190.0
		SD	39.8			89.9
2M	5	High	264.8	2F	5	219.6
		SD	75.8			98.7
3M	5	High	191.6	3F	5	291.6
		SD	48.6			224.7
4M	5	High	112.0*	4F	4	216.0
		SD	20.4			92.2
1M	5	Low	550.4	1F	5	600.6
		SD	175.0			206.2
2M	5	Low	634.0	2F	5	638.2
		SD	194.5			337.8
3M	5	Low	575.6	3F	5	634.8
		SD	37.5			143.5
4M	5	Low	418.6	4F	4	598.3
		SD	101.1			193.7

*p<0.05

Mean serum T4 concentrations (pg/mL)

 

Group	Dose (mg/kg bw)		F0 Males
1	0	Mean	40700
		SD	6130
		CV%	15.1
		N	10
2	5	Mean	46800
		SD	6410
		CV%	13.7
		N	10
3	15	Mean	50600
		SD	6390
		CV%	12.6
		N	10
4	40	Mean	49200
		SD	7100
		CV%	14.4
		N	9

 

Summary of treatment related findings in the epididymides and testes for animals killed after 5

weeks of treatment

Group		Control	5 mg/kg bw	15 mg/kg bw	40 mg/kg bw
Epididymides	No. examined	10	10	10	9
Cell Debris, Luminal	Minimal	0	0	1	7
	Total	0	0	1	7
Infiltrate, Inflammatory Cell	Minimal	0	0	0	2
	Moderate	0	0	0	1
	Total	0	0	0	3
Edema	Slight	0	0	0	1
	Total	0	0	0	1
Testes	No. examined	10	10	10	9
Degeneration/Atrophy, Tubular	Minimal	0	0	0	2
	Slight	0	0	0	2
	Total	0	0	0	4

 

Group 4 (40 mg/kg bw) Relationship between pregnancy rate and effects on testes and epididymides in males

male	female	epididymides	testes	pregnancy
1	1	+	+
2	2
3	3			+
4	4	+	+	+
5	5	+	+	+
6	6	+		+
7	7
8	8	+
9	9	+	+
10	10	+		+*

* one autolysed fetus was found

Applicant's summary and conclusion

Conclusions:

Oral administration of the substance was well tolerated in the adult animals but was associated with changes in the testes and epididymides of males treated at 40 mg/kg/day. 
The no-observed-adverse-effect-level (NOAEL) for systemic toxicity was considered to be 15 mg/kg/day.

Executive summary:

Three groups of ten male and ten female rats received the substance at doses of 5, 15 or 40 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, water, at the same volume-dose as treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis (T4), estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

Two animals died prematurely; these deaths were considered incidental and not related to treatment.

There were no treatment related adverse effects of treatment on clinical condition, sensory reactivity, grip strength, body weight gain, food intake, haematology, blood chemistry or organ weight measurements in males and females. 

The assessment of motor activity scores indicated that males receiving 40 mg/kg/day were slightly less active than the control males.

Estrous cyclicity was unaffected by the administration of the substance at all dose levels. Out of 10 females that mated in Group 4 (40 mg/kg/day), only five achieved pregnancy

Hematological examination for males revealed, when compared with controls, slightly low hematocrit counts attaining statistical significance at 15 or 40 mg/kg/day. A slight increase in mean cell volume (attaining statistical significance) was observed in females treated at 40 mg/kg/day. In addition a decrease of the number of lymphocytes was seen in females at 40 mg/kg bw.

Biochemical evaluation revealed no treatment-related findings.

At the macroscopic examination of the adult animals no treatment related effects were observed. Pale areas in heart and kidneys could be related to mineralization as observed during histopathological examination.

At the microscopic examination of the adult animals changes related to treatment with the substance were seen in males at 40 mg/kg bw. These findings included minimal to slight degeneration/atrophy in the testes in 4/9 males treated at 40 mg/kg/day. A minimal increase in luminal cell debris in the epididymides was seen in the majority of males treated at 40 mg/kg/day; this was also present in one animal given 15 mg/kg/day, but at this dose level the incidence and severity is within the historical control range (0 -20%). In three animals given 40 mg/kg/day, the luminal cell debris were associated with minimal to moderate interstitial inflammatory cell infiltrate. 

Oral administration of the substance was well tolerated in the adult animals but was associated with changes in the testes and epididymides of males treated at 40 mg/kg/day. A reduction in fertility at 40 mg/kg/day was also evident with only 5 / 10 females pregnant, however, there was no correlation between the findings in the testes and those females which were found not pregnant; as such it remains unclear if the reduced fertility must be attributed to the males or the females treated with the substance. Apart from the reduction in fertility, there was no systemic toxicity detected in females treated with the substance.

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity was considered to be 15 mg/kg/day.