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Toxicological information

Neurotoxicity

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Description of key information

Ziram caused reduction of neurotoxic esterase upon subchronic oral exposure and increased locomotor activity in the DNT study. The effects occurred at doses which caused general toxicity (impaired feed consumption and BW development).

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records
Reference
Endpoint:
neurotoxicity: oral
Remarks:
developmental
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Oct 2007 to 16 July 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP / guideline study without relevant deviations
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 426 (Developmental Neurotoxicity Study)
Deviations:
yes
Remarks:
assessments of learning and memory, motor and sensory function, and habituation were not conducted in this study. These parameters were already examined during the 2-generation study (Nemec, 1996) from the same laboratory using the same dose groups.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Age at study initiation: 81 days
- Weight at study initiation: 225 g to 288 g on GD0
- Fasting period before study: no
- Housing: After mating, the females were individually housed in plastic maternity cages with nesting material.
- Diet: PMI Nutrition International, LLC, Certified Rodent, LabDiet® 5002, ad libitum
- Water: Reverse osmosis-purified (on-site) drinking water, ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8°C to 22.3°C
- Humidity (%): 38.0% to 60.4%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 9 November 2007 To: 16 July 2009
Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002
- Storage temperature of food: refrigerated

DIETARY LEVELS
Actual target test substance concentrations were 60, 180 and 540 ppm.
Due to excessive maternal toxicity, the 540 ppm exposure level was lowered to 360 ppm beginning on lactation day 4 and continuing throughout the remainder of the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The assessments met the WIL Research Laboratories, LLC Standard Operating Procedures (SOP) acceptance criteria for test substance homogeneity, i.e., the variability for the mean concentration was ≤10% RSD at a concentration within the acceptable limits (85% to 115% of the fortified concentration), with the following exception. The formulation prepared at a fortified test substance concentration of 72 ppm and assessed for test substance homogeneity failed to meet the SOP requirement due to high intra-assay variability (24% RSD). However, the high RSD was the result of only 1 of 6 samples (first sample collected from the bottom strata was 45.3% of the fortified concentration whereas the second sample collected from the bottom strata was 95.0% of the fortified concentration). Therefore, the results of this analysis were considered acceptable.
Various assessments of test substance stability in formulations following storage at room temperature for 24 hours met the previously stated acceptance criteria with the following exceptions. The post-storage concentration in formulations fortified at a test substance concentration of 72 ppm (target concentration 60 ppm) and prepared on 26 October 2007 and 7 November 2007 was 75.9% and 61.4% of the target concentration, respectively. Also, the formulations prepared on 26 October 2007 and following refrigerated storage for 10 days met the stated acceptance criteria with the following exception. The post-storage concentration in the formulation fortified at a test substance concentration of 540 ppm (target concentration 540 ppm) was 82.1% of the target concentration. The low concentrations of the test substance detected in the test diet relative to the target concentrations following room temperature storage were consistent with previous studies (Nemec, 1996; WIL-223003); therefore, these results were considered acceptable.
Exposure formulations fortified at test substance concentrations of 0, 72, 207, 360, and 540 ppm offered to animals on study were analyzed to confirm test substance concentration, and the results met the previously mentioned SOP acceptance criteria for concentration acceptability in diet admix formulations, with the following exceptions.
The formulation prepared on 7 November 2007 at a fortified concentration of 72 ppm was 80.5% of the fortified concentration. This formulation was not introduced to the animals and was successfully reformulated on 9 November 2007. The formulation prepared on 14 November 2007 at a fortified concentration of 207 ppm (target concentration of 180 ppm) was 119% of the fortified concentration.
Duration of treatment / exposure:
GD6 to PND21
Frequency of treatment:
ad libitum
Remarks:
Doses / Concentrations:
5/8/12 mg/kg bw/day
Basis:
actual ingested
during gestation / LD1-4 / LD4-21
Remarks:
Doses / Concentrations:
13/23/34mg/kg bw/day
Basis:
actual ingested
during gestation / LD1-4 / LD4-21
Remarks:
Doses / Concentrations:
27/59/61 mg/kg bw/day
Basis:
actual ingested
during gestation / LD1-4 / LD4-21
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based on the 2-generastion study with ziram (Nemec, 1996, IUCLID Section 7.8.1)
Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Females expected to deliver were also observed twice
daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor or other difficulties).

BODY WEIGHT: Yes
Individual maternal body weights were measured on gestation days 0 and 6-20 (daily) and on lactation days 1-21 (daily). Group mean body weights were calculated for each of these days. Group mean body weight changes were calculated for each corresponding interval of gestation and lactation, and also for gestation days 6-9, 9-12, 12-15, 15-18, 18-20 and 6-20 and for lactation days 1-4, 4-7, 7-11, 11-14, 14-17, 17-21 and 1-21.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Individual maternal food consumption was recorded on gestation days 0 and 6-20 (daily) and on lactation days 1-21 (daily).

OPHTHALMOSCOPIC EXAMINATION: No
Specific biochemical examinations:
None
Neurobehavioural examinations performed and frequency:
FUNCTIONAL OBSERVATIONAL BATTERY: No

LOCOMOTOR ACTIVITY: Yes
- Locomotor activity testing was performed in a room equipped with a white-noise generation system set to operate at 70±10 decibels (db). Locomotor activity was assessed for 20 rats/sex/group on PND 13, 17, 21 and 61.
The same animals were tested at each interval. Locomotor activity was measured automatically using the Kinder Scientific Motor Monitor System (Kinder Scientific, LLC, Poway, California).
Each animal was tested separately. Data were collected in 5-minute epochs (print intervals) and the test session duration was 60 minutes.
Data for ambulatory and total locomotor activity were tabulated. Total locomotor activity was defined as a combination of fine locomotor skills (i.e., grooming; interruption of a single photobeam) and ambulatory locomotor activity (e.g., interruption of 2 or more consecutive photobeams).
Sacrifice and (histo)pathology:
OFFSPRING
- Time point of sacrifice of offspring selected for brain weight or neuropathological evaluation: postnatal day 21 or 72
- Description of postmortem examinations:
- At postnatal day 21 or 72, 15 pups/sex/group were macroscopically examined for neuropathology
- Brains from pups perfused on PND 21 and 72 were prepared for microscopic neuropathological examination (10 rats per sex). The brains were prepared for a qualitative histopathological
examination by embedding in paraffin, sectioning and staining with hematoxylin and eosin. Sections from all major brain regions (including olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, midbrain [tectum, tegmentum, cerebral peduncles, central gray matter], cerebellum, pons and medulla oblongata [a full coronal section was prepared]) were examined from the first 10 rats/sex in the control and high-exposure groups on PND 21 and 72, first 10 males in the low- and mid-exposure groups on PND 21 and the first 10 females in the low- and mid-exposure groups that met the criteria for measurement.

- Quantitative examinations of the brains from the selected PND 21 and PND 72 offspring included the following.
Specific levels analyzed, designated as Levels 1, 3 and 5, were defined as follows.
Level 1 was a coronal section of rostral cerebrum, including caudoputamen, Level 3 was a coronal section of mid-cerebrum (cerebral cortex, hippocampal formation, thalamus, etc.) and Level 5 was a mid-sagittal section of cerebellum and pons.
Measurements on Levels 1 and 3 were bilateral and were averaged for each animal.
Measurements on Level 5 were not paired since the other half of these tissues were sectioned transversely to enable visualization of cerebellar nuclei.
Statistics:
Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-exposed group to the control group by sex.

ONE-WAY ANOVA
Mean maternal and offspring body weights and body weight changes, maternal food consumption, gestation lengths, former implantation sites, unaccounted-for sites, numbers of pups born, live litter sizes, day of attainment of balanopreputial separation or vaginal patency, body weight on the day of attainment and brain weight, length, width and morphometric data

If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-exposed groups to the control group. Mean litter proportions (percent per litter) of pup viability and males per litter were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-exposed groups to the control group. Neuropathological data were analyzed using the Fisher’s Exact Test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
in the 540/360 ppm group
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
in the 540/360 ppm group
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Clinical biochemistry findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
enhanced locomotor activity in the 540/360 ppm group
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Other effects:
no effects observed
Details on results:
BODY WEIGHT AND WEIGHT GAIN
see Table 1 and 2.

FOOD CONSUMPTION AND COMPOUND INTAKE (IF FEEDING STUDY)
see Table 3 and 4.

NEUROBEHAVIOUR
see attached Figure for locomotor activity
Dose descriptor:
LOAEL
Effect level:
207 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increase in total and ambulatory counts and lower habituation to the test environment.
Remarks on result:
other: Generation: offspring (migrated information)
Dose descriptor:
LOAEL
Effect level:
34 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Dose received during PND4-21
Remarks on result:
other: Generation: offspring (migrated information)
Dose descriptor:
NOAEL
Effect level:
60 mg/kg diet
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Generation: offspring (migrated information)
Dose descriptor:
NOAEL
Effect level:
12 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Dose received during PND4-21
Remarks on result:
other: Generation: offspring (migrated information)
Dose descriptor:
LOAEL
Effect level:
207 mg/kg diet
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Reductions in mean maternal body weights, body weight gains and food consumption.
Remarks on result:
other: Generation: maternal (migrated information)
Dose descriptor:
LOAEL
Effect level:
13 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Dose received during gestation
Remarks on result:
other: Generation: maternal (migrated information)
Dose descriptor:
NOAEL
Effect level:
60 mg/kg diet
Based on:
test mat.
Sex:
female
Remarks on result:
other: Generation: maternal (migrated information)
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Dose received during gestation
Remarks on result:
other: Generation: maternal (migrated information)

Table 1: Summary of Body Weight Changes During Gestation [g]

 

0 ppm

72 ppm

207 ppm

540 / 260 ppm

DAY 0-6

MEAN

27.

30.

30.

25.

S.D.

7.4

7.2

7.8

9.2

N

25

25

25

23

DAY 6-7

MEAN

3.

0.

-7.**

-11.**

S.D.

5.5

5.8

5.4

6.2

N

25

25

25

23

DAY 7-8

MEAN

4.

6.

4.

-3.**

S.D.

4.4

4.5

3.9

4.3

N

25

25

25

23

DAY 8-9

MEAN

4.

6.

6.

-1.**

S.D.

5.0

5.4

5.4

3.4

N

25

25

25

23

DAY 9-10

MEAN

4.

4.

5.

1.**

S.D.

3.5

3.8

4.5

3.8

N

25

25

25

23

DAY 15-16

MEAN

7.

10.

11.

10.

S.D.

9.3

5.8

5.5

3.9

N

25

25

25

23

DAY 16-17

MEAN

17.

11.*

12.

13.

S.D.

9.5

8.4

3.5

5.2

N

25

25

25

23

DAY 17-18

MEAN

13.

18.*

16.

10.

S.D.

6.3

7.6

4.0

4.3

N

25

25

25

23

DAY 18-19

MEAN

17.

14.

13.*

13.**

S.D.

6.6

3.8

4.6

3.6

N

25

25

25

23

DAY 19-20

MEAN

16.

15.

13.*

10.**

S.D.

4.9

3.5

4.4

3.7

N

25

25

25

23

DAY 6-20

MEAN

115.

114.

97.**

58.**

S.D.

12.5

12.4

13.3

20.8

* = Significantly different from the control group at 0.05 using Dunnett's test

** = Significantly different from the control group at 0.01 using Dunnett's test

MEAN DIFFERENCES CALCULATED FROM INDIVIDUAL DIFFERENCES

NONGRAVID WEIGHT(S) NOT INCLUDED IN CALCULATION OF MEAN

 

Table 2: Summary of Body Weight Changes During Lactation [g]

 

0 ppm

72 ppm

207 ppm

540 / 260 ppm

DAY 1-4

MEAN

14.

13.

8.

9.

S.D.

10.2

10.0

9.4

10.0

N

25

25

25

23

DAY 4-7-A

MEAN

8.

9.

12.

26.**

S.D.

7.8

8.1

11.2

8.1

N

22

25

25

22

DAY 7-11

MEAN

16.

13.

15.

18.

S.D.

7.9

9.0

12.5

9.6

N

22

25

25

22

DAY 11-14

MEAN

10.

10.

11.

11.

S.D.

9.0

7.3

9.4

8.1

N

22

25

25

22

DAY 14-17

MEAN

0.

7.*

7.*

7.

S.D.

8.4

9.5

10.6

8.4

N

22

25

25

22

DAY 17-21

MEAN

-8.

-6.

-4.

3.**

S.D.

10.6

13.8

8.2

9.6

N

22

25

25

22

DAY 1-21

MEAN

41.

45.

50.

74.**

S.D.

14.8

11.6

13.4

15.0

** = Significantly different from the control group at 0.01 using Dunnett's test

MEAN DIFFERENCES CALCULATED FROM INDIVIDUAL DIFFERENCES

A = The 540 ppm test diet concentration was lowered to 360 ppm on lactation day 4.

Table 3: Summary of Food Consumption During Gestation [g/animal/day]

 

0 ppm

72 ppm

207 ppm

540 / 260 ppm

DAY 6-9

MEAN

20.

21.

17.**

9.**

S.D.

2.0

2.2

3.3

2.3

N

25

25

25

23

DAY 9-12

MEAN

21.

22.

20.

12.**

S.D.

2.2

1.8

2.0

3.0

N

25

25

25

23

DAY 12-15

MEAN

21.

23.*

21.

16.**

S.D.

2.2

1.9

2.4

3.0

N

25

25

25

23

DAY 15-18

MEAN

22.

24.*

23.

19.**

S.D.

3.0

2.3

2.8

2.8

N

25

25

25

23

DAY 18-20

MEAN

22.

23.

21.

18.**

S.D.

4.6

2.4

3.4

3.8

DAY 6-20

MEAN

21.

23.*

20.

14.**

S.D.

2.2

1.6

2.0

2.1

N

25

25

25

23

* = Significantly different from the control group at 0.05 using Dunnett's test

** = Significantly different from the control group at 0.01 using Dunnett's test

 

 

Table 4: Summary of Food Consumption During Lactation [g/animal/day]

 

0 ppm

72 ppm

207 ppm

540 / 260 ppm

DAY 1-4

MEAN

35.

36.

32.

29.**

S.D.

5.6

6.0

4.1

4.2

N

25

25

25

23

DAY 4-7-A

MEAN

39.

40.

39.

38.

S.D.

5.4

5.0

4.1

3.9

N

22

25

25

22

DAY 7-11

MEAN

50.

49.

48.

46.

S.D.

5.0

6.2

4.6

3.9

N

22

25

25

22

DAY 11-14

MEAN

60.

58.

57.

54.**

S.D.

5.5

6.4

5.5

4.4

N

22

25

25

22

DAY 14-17

MEAN

62.

63.

61.

60.

S.D.

5.8

7.3

4.3

3.9

N

22

25

25

22

DAY 17-21

MEAN

66.

65.

62.

64.

S.D.

8.2

7.3

5.2

4.0

N

22

25

25

22

DAY 1-21

MEAN

53.

52.

50.

49.*

S.D.

5.2

5.5

3.5

2.5

* = Significantly different from the control group at 0.05 using Dunnett's test

** = Significantly different from the control group at 0.01 using Dunnett's test

A = The 540 ppm test diet concentration was lowered to 360 ppm on lactation day 4.

Conclusions:
Ziram led to increases in offspring locomotor activity at doses that were maternally toxic. Ziram had no effects on litter paramters including the achievement of developmental landmarks and sexual maturation.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
12 mg/kg bw/day
Species:
rat
Quality of whole database:
excellent

Additional information

Justification for selection of effect on neurotoxicity via oral route endpoint:
most sensitive effect: increased offspring locomotor activity

Justification for classification or non-classification

Ziram caused behavioral effects only at doses that had caused maternal toxicity. Such non-specific effects do not qualify the substance for a respective classification.