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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 JULY 2012 to 23 JANUARY 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted in accordance with current test guidelines and GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): AZDN
- Physical state: White powder
- Analytical purity: 99%
- Purity test date: 08 August 2012
- Lot/batch No.: 7582
- Expiration date of the lot/batch: 31 March 2013
- Stability under test conditions: Stable at 0.1 and 15 mg/mL for 10-11 days under refrigerated conditions (2 to 8C)
- Storage condition of test material: 2 to 8C in the dark

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Margate, UK
- Age at study initiation: 26 to 30 days old on arrival, approximately 5-7 weeks old at start of dosing
- Weight at study initiation: Males = 127.2 to 192.3 g, Females = 111.6 to 167.7 g at start of dosing
- Fasting period before study: No
- Housing: Animals housed in groups of two, in cages that conform with the 'Code of practice for the housing and care of animals used in scientific procedures' (Home Office, London, 1989). Bedding provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester, UK)
- Diet (e.g. ad libitum): SQC Rat and Mouse Maintenance Diet No 1, Expanded, (Special Diets Services Ltd, Witham, UK) ad libitum
- Water (e.g. ad libitum): Mains water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24C
- Humidity (%): 45 to 65%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

IN-LIFE DATES: From: To: 04 September 2012 to 05 December 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in corn oil following dispensary SOPs and the formulation method 8266542_O_01D. The Group 2 formulation for Week 3 of dosing was prepared differently to formulation method 8266542_O_01D. The test substance was weighed into a small beaker (due to no available large capacity beaker compatible with the balance), which was not large enough to take the vehicle as well. The test substance beaker was rinsed out thoroughly with the vehicle and the test substance and rinsings were added to the vehicle.
The formulations were stored refrigerated (2 to 8C) in a sealed container, protected from the light.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil selected due to stability of test substance in the vehicle, and use in previous study R-95-007.
- Concentration in vehicle: 0.1 (low), 0.4 (intermediate) or 2.0 mg/mL (high)
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): MKBH894V
- Purity: Not available - on file with the manufacturer
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HOMOGENEITY: Samples (three aliquots from the top and three from the bottom of the test substance formulations and two random aliquots from control/vehicle formulations) were taken in Week 1 and 13 for analysis of homogeneity.
CONCENTRATION VERIFICATION: Samples (three aliquots from the top and three from the bottom of test substance formulations and two random aliquots from control/vehicle formulations) were taken in Week 1 and 13 for analysis of achieved concentration. The mean of the homogeneity results was taken as the achieved concentration where the sampling occasion coincided.
Duration of treatment / exposure:
92 days
Frequency of treatment:
Animals dose once daily vial oral gavage
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
10 mg/kg/day
Basis:
other: nominal in formulation
Remarks:
Doses / Concentrations:
2 mg/kg/day
Basis:
other: nominal in formulation
Remarks:
Doses / Concentrations:
0.5 mg/kg/day
Basis:
other: nominal in formulation
No. of animals per sex per dose:
10 male and 10 female animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: High dose (10 mg/kg/day) selected based on previously conducted study in this species (Combined Repeated Dose and Reproductive/Developmental Toxicity Screening Test of 2,2'-Azobis(2-methylpropionitrile) by Oral Administration in Rats; study number R-95-007).
Intermediate dose (2 mg/kg/day) was the approximate geometric mean between the low and high dose levels and was proposed in order to obtain a No Observed Adverse Effect Level (NOAEL) in the possible event that 10 mg/kg/day was not well tolerated after dosing. The low dose level (0.5 mg/kg/day) was proposed in order to try and obtain a NOEL.

- Rationale for animal assignment: Total randomisation procedure used to assign animals to treatment groups.
Positive control:
None used

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Beginning and end of the working day for signs of ill health or overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination at weekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights recorded on Day -7, on the first day of dosing, at weekly intervals and before necropsy.

FOOD CONSUMPTION:
The amount of food consumed by each cage of animals was determined weekly. Consumption was calculated as g/animal/day.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
Investigations performed on all animals pre-treatment and Group 1 and 4 animals in Week 12. A mydriatic agent was instilled into the eyes before examination.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were withdrawn from the jugular vein of all animals in Week 13.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: Haemoglobin, red blood cell (erythrocyte) count, haematocrit packed cell volume, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, reticulocyte count, red cell distribution width, haemoglobin ditribution width, total and differential white cell count, platelet count, platelet crit, mean platelet volume, platelet distribution width.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were withdrawn from the jugular vein of all animals in Week 13.
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total cholesterol, total bilirubin, total protein, albumin, globulin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorous, creatinine, urea, glucose.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected overnight from all animals in Week 12.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: Volume, colour, turbidity, specific gravity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, microscopy of sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: All animals subjected to a battery of behavioural tests and observations in Week -1 and once weekly to the end of the dosing period.
- Dose groups that were examined: All animals
- Battery of functions tested:
Weekly observations - before removal from the cage: posture, activity, gait, tremor, convulsion, excessive vocalisation, arousal upon opening cage.
Weekly observations - after removal from the cage: Ease of removal, ease of handling, excessive vocalisation, tremor, convulsion, palpebral closure, exophthalmus, lacrimation, lacrimation type, salivation, respiration, piloerection, appearance of fur, other.
Open field observations (each animal was placed into an open field arena for 2 minutes): Latency to first step, posture, arousal, circling, gait type, gait type severity, stereotypy, tremor, convulsion, other.
Open field tests: Approach response, touch response, tail pinch, air righting ability, pupillary response, corneal tactile response, auditory startle response, hindlimb foot splay, forelimb and hindlimb grip strength.

OTHER:
Locomotor activity: Assessed for each anmal in an automated photocell activity recorder for 30 minutes on one occasion during Week 13. Activity counts were recorded at 2-minute intervals. The following parameters were determined: Total activity, total mobile counts.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
Other examinations:
Immunhistochemical investigations: Additional sections of the kidney from all animals in Groups 1 to 4 were stained immunohistochemically for alpha-2-microglobulin. Immunohistochemical demonstration methods were used to demonstrate hyaline in the kidney. Histologically prepared sections of the kidney were immunostained using a commercial antibody to hyaline. Following incubation with the appropriate primary antibody, sections were then incubated with a suitable secondary biotinylated antibody and then visualised with a tertiary reagent and chromogen.
Statistics:
The control group (Group 1) was taken as the baseline group with which the treated groups (Groups 2, 3 and 4) were compared.
Body weight gains, food consumption, terminal body weights, haematology, clinical chemistry and urine analysis variables were analysed using one-way analysis of variance (ANOVA), separately for each sex. Levene's test for equality of variances among the groups was performed. Where this showed no evidence of heterogeneity (P>0.05), and the group effect from the ANOVA was significant (P=0.05), pairwise comparisons with control were made using Dunnett's test.
Where Levene’s test was significant (P=0.05), a rank-transformation was applied to the data prior to analysis.
No analysis was performed for variables with values above or below the limit of the assay.
Some variables were not analysed due to there being too few values for meaningful analysis.
Organ weights were analysed using Analysis of Covariance (ANCOVA), for each sex separately, using the terminal body weight as the covariate. This analysis depends on the assumption that the relationship between the organ weights and the covariate is the same for all groups and the validity of this assumption was tested. Where there was no evidence of heterogeneity among the slopes (P>0.01), and the group effect from the ANCOVA was significant (P=0.05), pairwise comparisons with control were made using Dunnett's test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were six decedents during the study. Animal 5M (control) died on Day 42, Animal 48F (control) died on Day 72, Animal 13M (given 0.5 mg/kg/day) died on Day 73, Animal 51F (given 0.5 mg/kg/day) died on Day 75, and Animal 1M (control) died on Day 84 of the study. In all these animals there were various inflammatory lesions in the thoracic cavity, including lung pleuritis, heart pericarditis and epicarditis, and serositis around the thymus and aorta. These findings were suggestive of death due to dosing errors and subsequent inflammatory changes, and were considered to be incidental deaths due to study procedures. For Animal 11M (given 0.5 mg/kg/day) the cause of demise was not determined. There were no deaths considered to be related to effects of AZDN. In addition to findings associated with terminal events, the range of macroscopic and microscopic findings in decedents was generally similar to those in animals surviving to terminal kill. For clinical signs, there were considered to be no effects of treatment. Clinical signs were observed but the type, severity and distribution were typical of rats of this age and strain.
Mortality:
no mortality observed
Description (incidence):
There were six decedents during the study. Animal 5M (control) died on Day 42, Animal 48F (control) died on Day 72, Animal 13M (given 0.5 mg/kg/day) died on Day 73, Animal 51F (given 0.5 mg/kg/day) died on Day 75, and Animal 1M (control) died on Day 84 of the study. In all these animals there were various inflammatory lesions in the thoracic cavity, including lung pleuritis, heart pericarditis and epicarditis, and serositis around the thymus and aorta. These findings were suggestive of death due to dosing errors and subsequent inflammatory changes, and were considered to be incidental deaths due to study procedures. For Animal 11M (given 0.5 mg/kg/day) the cause of demise was not determined. There were no deaths considered to be related to effects of AZDN. In addition to findings associated with terminal events, the range of macroscopic and microscopic findings in decedents was generally similar to those in animals surviving to terminal kill. For clinical signs, there were considered to be no effects of treatment. Clinical signs were observed but the type, severity and distribution were typical of rats of this age and strain.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Males treated with 10 mg/kg/day showed a slight reduction in bodyweights at the end of the treatment period (-5.2% compared with controls) but differences were not statistically significant. There were no similar differences in females.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects on food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
Changes not considered to be of toxicological significance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Changes not considered to be of toxicological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Minor changes in urine parameters of males at all dose levels. Males at 10 mg/kg/day also had a paler colour urine and although there was a slight increase in volume, this was not statistically significant.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were considered to be no effects of treatment on any parameter of the Functional Obersvation Battery.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were treatment-related changes in liver and kidney weights when compared with concurrent controls, and these changes correlated with findings seen microscopically.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A higher level of pale and/or mottled kidneys recorded in all treated male groups. No other macroscopic findings considered related to treatment with AZDN. Findings related to treatment with AZDN were noted in the liver (both sexes) and in male kidneys.
Details on results:
ORGAN WEIGHTS:
Group mean adjusted liver weights were increased in all treated male groups (M: 8, 4 and 9%, respectively) and in females given 2 or 10 mg/kg/day (F: 9 and 23%, respectively). Equivalent liver to body weight ratios were also increased (M: 7, 2 and 10%, F: 8 and 24%, respectively) as were liver to brain weight ratios (M: 6, 4 and 4%, F: 9 and 20%, respectively) when compared with concurrent controls.
Group mean adjusted kidney weights were increased in all treated male groups (M: 9, 21 and 20%, respectively). Equivalent kidney to body weight ratios were also increased (M: 8, 19 and 20%, respectively) as were kidney to brain weight ratios (M: 7, 21 and 14%, respectively) when compared with concurrent controls. Kidney weights in treated females were similar to controls.
Thymus weights in treated males were generally decreased compared with concurrent controls, but in females thymus weights were increased compared with concurrent controls. As there was no microscopic correlation with these findings, and inconsistency between the sexes, these organ weight changes in the thymus were not considered to be related to treatment with AZDN.
All other organ weight and/or organ weight ratio changes, including those that were statistically significant, were attributed to normal biological variation and were considered not to be test-item related as they were either small in magnitude, not dose-dependent, inconsistent between the sexes, due to normal inter-animal variability and/or lacked a histopathological correlate.

Effect levels

Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Macroscopic and Microscopic Observations

In the liver, hepatocyte hypertrophy was seen in all treated male groups and in females given 2 mg/kg/day (7 females grade 1, 1 female grade 2) or 10 mg/kg/day (10 females grade 2), and showed a dose-related trend. The finding correlated with the increase in liver weights noted at necropsy, and was characterised by an increase in size of hepatocytes, mainly in the centrilobular areas, with paler eosinophilic cytoplasm. In treated males there were also minor levels of hepatocyte vacuolation. This finding was characterised by generally large clear vacuoles in hepatocytes.

Incidence of hepatocyte hypertrophy and hepatocyte vacuolation: liver – terminal kill

 

 

Males

 

Females

 

 

1M

2M

3M

4M

 

1F

2F

3F

4F

Tissue and finding

Level (mg/kg/day)

0

0.5

2

10

 

0

0.5

2

10

 

 

 

 

 

 

 

 

 

 

 

Liver

No. examined

8

8

10

10

 

9

9

10

10

hepatocyte hypertrophy

Grade -

8

4

0

0

 

9

9

2

0

 

1

0

2

0

0

 

0

0

7

0

 

2

0

2

9

3

 

0

0

1

10

 

3

0

0

1

7

 

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

hepatocyte vacuolation

Grade -

8

7

1

5

 

9

9

10

10

 

1

0

1

9

5

 

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

Key: “-“ =finding not present, 1 = minimal, 2 = slight, 3 = moderate.


In the kidney there were higher levels of hyaline droplets and tubular basophilia/focal nephropathy in all treated male groups compared with controls, and levels of the findings were similar in all three treated groups. These findings correlated with the increase in kidney weights and the pale and/or mottled kidneys noted at necropsy. Hyaline droplets were characterised by bright eosinophilic inclusions in the proximal tubular cells of the kidney. Tubular basophilia/focal nephropathy was characterised by groups of tubules with basophilic cytoplasm, degenerating and regenerating tubules, sometimes with dilated tubules, granular casts and inflammatory cell infiltrate.

Hyaline droplets were not seen in females and the levels of tubular basophilia/focal nephropathy in females given 10 mg/kg/day were similar to controls.

The immunohistochemical staining for alpha 2u globulin showed some background staining, possibly due to cross-reactivity of the antibody, but levels of staining above the background, localised in the proximal convoluted tubules, generally correlated with the levels of hyaline droplets seen in males. Therefore the hyaline droplets were considered to be due to accumulation of alpha 2u globulin.

Incidence of hyaline droplets and tubular basophilia/focal nephropathy: kidney – terminal kill

 

 

Males

 

Females

 

 

1M

2M

3M

4M

 

1F

2F

3F

4F

Tissue and finding

Level (mg/kg/day)

0

0.5

2

10

 

0

0.5

2

10

 

 

 

 

 

 

 

 

 

 

 

Kidney

No. examined

8

8

10

10

 

9

2

1

10

 hyaline droplets

Grade -

0

0

0

0

 

9

2

1

10

 

1

2

0

0

0

 

0

0

0

0

 

2

6

0

0

0

 

0

0

0

0

 

3

0

8

10

10

 

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

tubular basophilia/focal nephropathy

Grade -

6

0

0

0

 

8

1

1

9

1

2

0

1

1

 

1

1

0

1

 

2

0

5

3

6

 

0

0

0

0

 

3

0

3

6

3

 

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

Key: “-“ =finding not present, 1 = minimal, 2 = slight, 3 = moderate.

 

There were no other microscopic findings related to treatment with/administration of AZDN.

Microscopic findings in other tissues were generally infrequent, of a minor nature and consistent with the usual pattern of findings in rats of this strain and age.

Applicant's summary and conclusion

Conclusions:
AZDN was well tolerated with minimal reaction to treatment and no evidence of neurotoxicity in either functional observation battery or locomotor activity. The NOAEL was considered to be 10 mg/kg/day.
Executive summary:

The objective of the study was to determine the toxicity of the test article, AZDN (2,2’-dimethyl-2,2’-azidopropionitrile), following daily oral (gavage) administration to the rat for 13 weeks.

The study was conducted at the request of the regulatory authorities in order to fulfill REACH requirements.

The oral route of administration was chosen because it is a likely human exposure route.

Groups ofratsof the Crl:CD(SD) strain were dosed as follows:

 

 

Number of Animals

Dose Level

Dose Concentration

Group

Subgroup

Male

Female

(mg/kg/day)

(mg/mL)

 

 

 

 

 

 

1 (Control)a

1 (Toxicity)

10

10

0

0

2 (Low)

1 (Toxicity)

10

10

0.5

0.1

3 (Intermediate)

1 (Toxicity)

10

10

2

0.4

4 (High)

1 (Toxicity)

10

10

10

2.0

 

 

 

 

 

 

a Group 1 received vehicle control article only

 

Assessment of toxicity was based on clinical signs, post dosing observations, functional observation battery and locomotor activity, body weights, food consumption, ophthalmoscopy, clinical pathology, organ weights and macroscopic and microscopic histopathology.

Incidental deaths due to study procedures were seen in controls and animals given 0.5 mg/kg/day, but there were no decedents during the study related to treatment with AZDN.

There were no clinical signs related to treatment and post dosing observations were consistent with reactions to taste of dosed materials. There were no toxicologically significant effects of treatment on ophthalmoscopy, functional observation battery, locomotor activity, bodyweights, food consumption or haematology.

There was a treatment related increase in creatinine in males. Females had higher calcium and lower chloride at all dose levels.

There were minor changes in urine parameters of males at all dose levels including reductions of urobilinogen and bilirubin scores, and increased amorphous debris scores. Males treated with 10 mg/kg/day also had paler colour urine and increased volumes.

Group mean adjusted liver weights were increased in all treated male groups and in females given 2 or 10 mg/kg/day. Group mean adjusted kidney weights were increased in all treated male groups. Kidney weights in treated females were similar to controls.

On macroscopic examination, a higher level of pale and/or mottled kidneys was recorded in all treated male groups.

On microscopic examination, hepatocyte hypertrophy was seen in the liver of all treated male groups and in females given 2 or 10 mg/kg/day, and showed a dose-related trend. There were also minor levels of hepatocyte vacuolation in the liver of treated males. In the kidney there were higher levels of hyaline droplets and tubular basophilia/focal nephropathy in all treated male groups compared with controls, and levels of the findings were similar in all three treated groups. Immunohistochemical staining confirmed the presence of alpha 2 microglobulin and therefore the hyaline droplets were considered to be due to accumulation of alpha 2u globulin.

In conclusion, daily administration of AZDN for 13 weeks at dose levels of 0.5, 2 or 10 mg/kg/day was well tolerated. There was no evidence of neurotoxicity. There were slight changes in some blood clinical chemistry and urine parameters at all dose levels. The liver and kidney were identified as target organs based on increased weights (at all dose levels in males) and microscopic findings of hepatocyte hypertrophy (at all dose levels in males and at =2 mg/kg/day in females) and focal nephropathy/tubular basophilia and hyaline droplets in males at all dose levels. The liver hypertrophy is considered an adaptive change commonly associated with metabolic burden and the changes in the kidney are considered specific to the male rat as confirmed by immunohistochemical staining. Therefore, the nature of these histopathological changes are considered to be of no toxicological relevance and the No-Observed-Adverse-Effect-Level (NOAEL) is considered to be 10 mg/kg/day.