3-iodo-2-propynyl butylcarbamate

Carcinogenicity

Description of key information

Based on data of two carcinogenicity studies applying the test item via oral route to mice and rats, it can be concluded that the test item is not considered to be carcinogenic.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989-06-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-2 (Carcinogenicity)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: P-2848-8603-P100

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: ca 4 weeks at arrival
- Weight at study initiation: ca. 21 g (males); ca. 18 g (females)
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 +/- 2 °C
- Humidity: 55 +/- 10 %
- Air changes: 15 - 20 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Diets were prepared by direct admixture of the test material to the diet and mixing for 20 min on a Winkworth Change Drum Mixer.

DIET PREPARATION
- Rate of preparation of diet: twice each week

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

100 g sample of diet from each group/sex was retained immediately after each diet preparation.
In addition, samples were analysed from all diets prepared for Weeks 1, 4, 13, 14, 18, 20, 23, 43, 52, 70, 71 and 78 of the study. Samples were analysed under IRI Project No. 336802.

Achieved dosages were generally close to nominal with the largest deviations being -2 %.
Analyses of formulated diets showed that they were generally acceptable (within +/- 15 % of nominal). There were occasional deviations from +/- 15% but these were isolated incidences and did not reflect any problem with the accuracy or precision of formulation.

Duration of treatment / exposure:

78 weeks

Frequency of treatment:

daily

Post exposure period:

not applicable

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: on the basis of results of a dose range finding study
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: not applicable

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

DERMAL IRRITATION: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly until week 13 and at 4 weekly intervals thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: monitored by visual inspection throughout the treatment period

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 51 and 77 weeks of dosing
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals of control and high dose

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 77 weeks of dosing
- How many animals: 20 males and 20 females randomly chosen from each treatment group
- Parameters examined: orbital sinus for measurement of plasma, red blood cell and brain cholinsesterase activity

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Adrenals; Aortic arch; Any abnormal tissue; Bladder; Bone (sternum and rib); Brain; Eyes; Heart; Intestine: duodenum, jejunum, ileum, caecum, colon, rectum; Kidneys; Liver (with gall bladder); Lungs (perfused); Mammary gland; Mesenteric lymph node; Muscle (thigh); Nasal Cavity; Oesophagus; Ovaries (with fallopian tubes); Pancreas; Pituitary; Prostate; Sciatic nerve; Seminal vesicles; Skin Spinal cord (cervical, thoracic and lumbar portions); Spleen; Stomach (glandular and non-glandular); Submaxillary salivary gland; Submandibular lymph node; Testes (plus epididymides); Thymus; Thyroids (with parathyroids examined where present); Tongue; Trachea; Vagina; Uterus

HISTOPATHOLOGY: Yes for control and high dose animals
Adrenals; Aortic arch; Any abnormal tissue; Bladder; Bone (sternum and rib); Brain; Eyes; Heart; Intestine: duodenum, jejunum, ileum, caecum, colon, rectum; Kidneys; Liver (with gall bladder); Lungs (perfused); Mammary gland; Mesenteric lymph node; Muscle (thigh); Nasal Cavity; Oesophagus; Ovaries (with fallopian tubes); Pancreas; Pituitary; Prostate; Sciatic nerve; Seminal vesicles; Skin Spinal cord (cervical, thoracic and lumbar portions); Spleen; Stomach (glandular and non-glandular); Submaxillary salivary gland; Submandibular lymph node; Testes (plus epididymides); Thymus; Thyroids (with parathyroids examined where present); Tongue; Trachea; Vagina; Uterus

Statistics:

Differences in survival between the Control and groups receiving test item were assessed graphically using Kaplan-Meier plots and tested formally using the Gehan-Wilcoxon test. As no notable differences were evident lesion incidences were analysed using Fisher's Exact Probability Test, two-tailed, on the findings which appeared to be biologically meaningful, one-tailed on tumour incidences. Body weight data were statistically analysed for homogeneity of variance using the F-max test. If the group variances appeared homogeneous a parametric ANOVA was used and pairwise comparisons made via Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous log or square root transformations were used in an attempt to stabilise the variances. If the variances were still heterogeneous then a non-parametric test such as Kruskal-Wallis ANOVA was used and pairwise comparisons made via Dunn Z test where considered appropriate.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs that could be attributed to dosing with the test item. Those observations recorded, such as perigenital swellings, encrustations around eyes and nose, staining on the body, are considered to be normal for mice of this age and strain.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

After statistical analysis there was no evidence of differential mortality patterns, nor was any common cause of death noted.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males
There was a marked reduction in body weight gain in the High dose group (23%). In terms of absolute body weight, the High dose group showed statistically significant differences (P<0.01) during Weeks 60-72 only.

Females
There was a moderate reduction in body weight gain in the High dose group (20%). In terms of absolute body weight, the High dose group showed statistically significant differences (P<0.05 - P<0.01) intermitantly throughout the dosing period.

There were no other notable intergroup differences.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males
Although there were slight reductions in food consumption (6% Low dose, 5% Intermediate dose and 7% High dose) these reductions are considered to be in the bounds of normal variability due to their magnitude and lack of relationship to dose level.

Females
All dose groups which received the test item showed food consumption slightly greater than that of Controls. These slight differences are, however, considered to be within the bounds of normal variability due to their magnitude.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

There was no visual intergroup difference in either sex.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Week 52 and 78:
There were no notable intergroup differences in either sex.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no notable intergroup differences in either sex.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were 2 macroscopic findings, one in females and one in males, which appeared to relate to treatment.
In the female High dose group there was a reduction in the incidence of bilateral renal pallor.
In the male High dose group there was an increased incidence of thyroid enlargement.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were a number of findings that were treatment related.
These included, as an increase in incidence compared to Control values, pneumonitis in the male High dose group, atrophic follicular vacuolation, generalised follicular enlargement and follicle coalescence in the thyroids of all males which received test item; renal pelvic epithelial inflammation in the female High dose group, atrophic thyroid follicular vacuolation in all females which received test item, generalised thyroid follicular enlargement in female Intermediate and High dose groups, thyroid follicle coalescence in the female Low dose group and chronic thyroid follicular inflammation in the female High dose group.
There was one treatment related finding which appeared to diminish in incidence with increasing dose level. This was hepatocytic periacinar vacuolation in the male High dose group.
There were a number of findings which were statistically significant which are believed to have been due to chance and did not reflect a true effect of treatment. These isolated significant incidences lacked dosage relationship with incidences at the High dose level. Such non treatment related findings included renal cortex chronic inflammation in the male Intermediate dose group, and renal cortex chronic inflammation in the female Low dose group.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was a single incidence of neoplasia that was associated with treatment. This was hepatocytic adenoma in the male High dose group. The incidence of 11 out of 50 is higher than that seen in comparable Control groups in recent studies carried out at IRI where the range of this finding was 1-8 out of 50.

Other effects:

no effects observed

Description (incidence and severity):

Predominant Pathology
There were no differences in the incidences of the subjectively recorded 'predominant pathology'. As is commonplace with this strain, the majority of animals had no predominant pathological changes. Common recorded changes occurred as pulmonary and hepatic neoplasia, especially in male mice, and reproductive tract disease of female mice.

Other changes that were recorded are those that are commonplace in this strain of laboratory rodent undergoing a toxicity trial of prolonged duration. Their incidence and degree were unremarkable and they were unaffected by treatment.

Key result

Dose descriptor:

LOEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Table 1 Summary of Results of the 78-Week Carcinogenicity Study in Mice

 

Parameter	Dose level [mg/kg bw/day]
	0		20		50		150		dose-response + /-
	m	f	m	f	m	f	m	f	m	f
Number of animals in group	50	50	50	50	50	50	50	50
Mortality	9	10	15	11	8	17	11	6	-	-
clinical signs	ne	ne	ne	ne	ne	ne	ne	ne	-	-
body weight gain (week 0 to 78) [g]	15.9	12.9	15.5	12.0	14.4	13.0	12.3	10.3	+	+
food consumption	ne	ne	ne	ne	ne	ne	ne	ne	-	-
water consumption	ne	ne	ne	ne	ne	ne	ne	ne	-	-
Plasma cholinesterase	ne	ne	ne	ne	ne	ne	ne	ne	-	-
RBC cholinesterase	ne	ne	ne	ne	ne	ne	ne	ne	-	-
Brain cholinesterase	ne	ne	ne	ne	ne	ne	ne	ne	-	-
haematology	ne	ne	ne	ne	ne	ne	ne	ne	-	-
Thyroid
enlarged	1	0	4	2	3	4	9	4	+	-
histopathology
Thyroid 1
number of organs examined	48		49		50		49
atrophic follicular vacuolation	0	0	26***	18***	37***	33***	28***	24***	+	+
follicle coalescence	3	5	19***	14*	16**	5	10	5	-	-
gen. follicular enlargement	1	2	14***	7	21***	13**	29***	27***	+	+
chronic inflammation	0	0	0	4	4	0	2	3	-	-
Thyroid 2
number of organs examined	47		46		49		50
atrophic follicular vacuolation	0	0	27***	19***	36***	27***	26***	30***	+	+
follicle coalescence	1	1	23***	10**	13**	5	8*	5	-	-
gen. follicular enlargement	2	2	17***	6	21***	13**	26***	27***	+	+
chronic inflammation	0	0	0	1	3	3	4	6	-	-
Liver
number of organ examined	50	50	50	50	50	49	50	50
inflammation	3	13	6	19	7	16	1	8	-	-
clear cell focus	1	0	1	0	1	1	0	0	-	-
periacinar hypertrophy	4	2	2	0	1	1	2	0	-	-
increased mitotic figures	2	1	0	0	1	0	1	1	-	-
focal hepatocytic hyperplasia	1	1	8	0	6	1	1	0	-	-
coagulative necrosis	0	3	2	2	1	3	2	0	-	-
focal hepatocytic hypertrophy	4	0	0	0	2	1	1	0	-	-
periacinar vacuolation	19	6	14	2	12	3	4***	5	+	-
centriacinar vacuolation	0	0	0	0	0	0	1	0	-	-
single cell necrosis	1	0	0	0	0	0	0	0	-	-
haemangioma	1	0	0	0	0	0	0	0	-	-
hepatocytic adenomaa)	4	0	3	1	5	1	11*	1	-	-
hepatocytic carcinoma	3	0	3	0	3	0	4	0	-	-
Kidney 1
both pale	0	7	3	4	2	5	1
number of organs examined	50	50	50	50	50	49	50	50
pelvic epithelial inflammation	16	14	15	24	23	20	16	26*
cortex chronic inflammation	2	8	6	18*	11	16	9	13
Kidney 2
number of organs examined	50	50	50	50	50	49	50	50
pelvic epithelial inflammation	9	8	9	13	17	15	10	12	-	-
cortex chronic inflammation	4	8	5	9	5	8	7	6	-	-
Lungs
number of organs examined	50	0	50	0	50	0	50	0
pneumonitis	1	1	2	1	4	3	9*	5	+	-

 

*          statistically significantly different from control, p<0.05

**         statistically significantly different from control, p<0.01

***       statistically significantly different from control, p<0.001

^a)         historical control data: incidence in hepatocellular adenoma: 1 to 8/50

 

Conclusions:

Dosing CD-I mice for 78 weeks with the test item resulted in findings in body weight gain (reductions in both sexes at 150 mg/kg bw/d) and histopathology (hepatic adenoma in males at 150 mg/kg bw/d and non neoplastic thyroid changes in males and females at 20, 50 and 150 mg/kg bw/d). A subsequent independent evaluation of these findings confirmed, that under the conditions of this study, the test item is not considered hepatocarcinogenic.

Executive summary:

Groups of 50 male and 50 female CD-I mice were dosed with the test item via the diet at concentrations calculated to achieve dose levels of 20 (Low dose), 50 (Intermediate dose) and 150 (High dose) mg/kg bw/d. A further group of 50 males and 50 females received untreated diet to act as Controls. During Weeks 52 and 78 blood samples were taken from all animals via a tailsnip and a differential blood smear prepared. A blood sample was also taken via the orbital sinus during Week 78 which was used for clinical chemistry. After 78 weeks of dosing all remaining animals were killed by carbon dioxide asphyxiation followed by exsanguination, followed by a gross necropsy. Histopathological evaluation was carried out.
There were no notable intergroup differences in either sex regarding mortality. There were no notable intergroup differences at clinical signs parameters. There was a moderate reduction in body weight gain in the female High dose group and a marked reduction in the male High dose group. However, in regards to food consumption, there were no notable intergroup differences. There were no notable intergroup differences in neither differential blood count nor clinical chemistry parameters.
AT gross pathological examination there was an increased incidence of thyroid enlargement in the male High dose group. Histopathological investigation revealed an increased incidence of hepatic adenoma in the male High dose group. There were unusual changes in thyroids which were thought to be atrophic and were not thought to be proliferative. They comprised atrophic follicular vacuolation, generalised follicular enlargement, follicle coalescence and chronic follicular inflammation seen singly or in some combination in all dose groups.

Subsequently, an independent panel review of the livers by another working group was performed. Evaluation of liver sections resulted in the conclusion that the test item given to male and female CD-I mice in the diet at levels of 20, 50, or 150 mg/kg bw/d for 78 weeks did not result in any carcinogenic effect. The incidence of hepatocellular adenomas in high dose males was increased over controls. However, this increase was not considered to be biologically relevant as there was no increase in the incidence of hepatocellular carcinomas, foci of cellular alteration, or combined benign and malignant hepatocellular tumors. There was no increase in the incidence of hepatocellular tumors or foci of cellular alteration in female mice from any treatment group.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989-03-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Principles of method if other than guideline:

Deviations:
- individual body weight of the animals designated for the interim necropsy after one year are not given
- hamatology, clinical chemistry, and urinalysis were not performed after 18 months. Only 10 instead of 20 animals per sex and group were bleed

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: P-2848-8603-P100

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (U.K.) Limited, Manston, Kent, U.K.
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: 191 - 195 g (males); 132 - 137 g (females)
- Housing: group housing: 5 males or 5 females per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 +/- 2 °C
- Humidity: 55 +/- 5 %
- Air changes: ca. 14 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Diets were prepared by direct admixture of the test material to the diet and mixing for 20 min on a Winkworth Change Drum Mixer.

DIET PREPARATION
- Rate of preparation of diet: twice each week

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A 100 g sample of diet from each group/sex was retained immediately after each diet preparation.
In addition, samples from all diets were analysed during Weeks 1, 3, 5, 7, 9, 12, 15, 18, 22, 23, 26, 27, 32, 33, 39, 45, 46, 51, 52, 57, 60, 64, 71, 77, 83, 91, 92 and 97 under a separate project (IRI Project No. 335018).

Duration of treatment / exposure:

Interim kill animals: 13, 26, 51 weeks
Complete study: 104 weeks

Frequency of treatment:

daily

Post exposure period:

not applicable

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

80 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Complete study: 50

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: preliminary sub-chronic toxicity study
- Rationale for animal assignment: random

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily examination for reaction to treatment; detailed clinical examination and palpation once each week

BODY WEIGHT: Yes
- Time schedule for examinations: weekly until week 13; and at 4 weely intervals after week 16

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, per group of n = 5; twice a week until week 13 and 4 weekly intervals thereafter

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: water consumption was monitored by visual inspection throughout the treatment period

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and at interim kill
- Dose groups that were examined: control and high-dose group (80 mg/kg bw/d)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13, 26 and 51 (interim kill group) and week 100
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 10 per sex per dose
- Parameters examined: Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, Hepato Quick test
- Differential blood count: parameters: neutrohpiles, lymphocytes, monocytes, and eosinophiles

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13, 26 and 51 (interim kill group) and week 100
- Animals fasted: Not specified
- How many animals: 10 per sex and dose
- Parameters examined: Blood urea nitrogen, glucose, AST, ALT, AP, sodium, potassium, calcium, chloride, total protein, albumin, albumin-globulin ratio, creatinine, inorganic phosphorus, bilirubin total, cholesterol, plasma RBC and brain cholinesterase activity using the methods of Ellman or Pilz

URINALYSIS: Yes
- Time schedule for collection of urine: week 13, 26 and 51 (interim kill group) and week 100
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters examined: Volume, pH, specific gravity, protein, glucose, ketones, blood pigments, bilirubin, urobilinogen; microscopic examination of the spun deposit

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes; all organs taken from control and high dose animals, as well as premature decedents from low- and mid- dose

Statistics:

Organ weight and body weight data were statistically analysed for homogeneity of variance using the F-max test. If the group variances appeared homogeneous a parametric ANOVA was used and pairwise comparisons made yia Student's t-test using Fisher's F-protected LSD. If the variances were non-homogeneous log or square root transformations were used in an attempt to stabilise the variances. If the variances remained non-homogeneous, then a non-parametric test such as a Kruskal-Wallis ANOVA was used.
Histopathology data were analysed using Fisher's Exact Probability test.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs that could be attributed to dosing with the test item. Those observations recorded, such as perigenital swellings, encrustations around eyes and nose, staining on the body, are considered to be normal for rats of this age and strain.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were 142 unscheduled deaths distributed as throughout the dose groups. There was no statistical evidence of any differentrial mortality between any of the dose groups receiving test item and controls nor was there any common factor evident.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males
There was a dose related reduction in body weight gain across all the dose groups which received test item; slight in the Low dose (11%); moderate in the Intermediate dose (14%) and marked in the High dose (29%). These reductions, in terms of absolute body weight, attained statistical significance (P < 0.05 – P < 0.001) from Week ca. 1 onwards.

Females
There was a moderate reduction in body weight gain seen in the Intermediate dose group (11%) and a marked reduction in the High dose group (24%). These changes in body weight attained statistical significance (P<0.05 - P<0.001) from Week ca. 1 onwards in terms of absolute body weight.
Body weight gain in the Low dose was similar to that of Controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males
There were slight reductions in total food consumed in all dose groups which received test item; 5% Low dose; 6% Intermediate dose and 10%, High dose group. Only the latter is considered to be outside the normal intergroup variability of food consumption.

Females
There were slight reductions in total food consumed in the Intermediate and High dose groups (6% and 4% respectively), but these are considered to be within the bounds of normality. Total food consumed in the Low dose was similar to that of Controls.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

Males
Week 13: The High dose group showed reductions compared to the Controls in haemoglobin concentration (7 %, P < 0.01), RBC counts (6 %, P < 0.01) and haematocrit (5 %, P < 0.05). The Low and Intermediate dose groups tended to have values close to the Controls. MCHC was also reduced in the Intermediate (3 %, P < 0.001) and High (3 %, P < 0.01) dose groups.

Week 26: The reductions in RBC parameters seen at Week 13 were not evident. The only instances of statistical significance were equivocal reductions in lymphocyte counts in the Intermediate (23 %, P < 0.05) and High (20 %, P < 0.05) dose groups, but the low magnitude of effect suggests this is of no importance biologically.

Week 51: As in Week 26 lymphocyte counts showed equivocal reductions in the Intermediate (20 %, P < 0.05) and High (17 %, not significant) dose groups, again not considered to be of biological importance.

Week 100 (Carcinogenicity Study Animals): All groups receiving test item showed decreased MCH values compared to Controls, but this is thought to be due to high Control values as there was no effect on the other RBC parameters. Platelet counts were increased compared to Controls in all groups receiving test item, but this is considered to be a chance effect due to lack of a relationship to dose level and high individual variability. Low dose neutrophil counts were decreased but this again did not follow a dose relationship.

Females
Week 13: The only instance of statistical significance was a slight increase in lymphocyte levels (36 %, P < 0.05) in the Low dose group, but this is considered a chance effect due to the small magnitude of effect and lack of an obvious pattern related to dose administered.

Week 26: Only the Low dose group Hepato Quick time showed a statistically significant effect (8 %, P < 0.05), but this is considered a chance effect due to the lack of effect in the other dose groups.

Week 51: On this occasion all groups receiving test item showed decreased MCH and MCV values compared to Controls, but this is thought to be due to high Control values as there was no effect on the other RBC parameters and there had been no difference at the earlier timepoints.

Week 100 (Carcinogenicity Study Animals): There was no evidence of any treatment related effects. Those intergroup differences that did occur showed no dose relationship.

In conclusion, haematological parameters were not considered to be changed by test item treatment. Because dose-response relationship could not be shown, effects were not consistent throughout the treatement period and/or were of low magnitude. Therefore, changes observed were considered within normal biological variation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Males
Week 13: The Intermediate and High dose groups ALT levels were lower than Controls with the reduction in the High dose group achieving statistical significance (22 %, P < 0.01). All groups receiving test item showed higher cholesterol levels than Controls, with the Low (16 %, P < 0.05) and High (21 %, P < 0.01) dose groups achieving statistical significance. Low dose group AP levels were lower (22 %, P < 0.01) than Controls, but is considered a chance effect as the other groups receiving test item showed no difference. There was a reduction (21 %, not statistically significant) in red blood cell cholinesterase activity in High dose rats.

Week 26: The Intermediate and High dose groups showed lower AST (20%, P<0.01 and 23%, P<0.001 respectively) and ALT (17%, P<0.05 and 24%, P<0.01 respctively) than Controls. As at Week 13 all groups receiving test item showed higher cholesterol levels than Controls, but only the High dose group achieved statistical significance (25%, P<0.001). Again, there was a reduction (20%, not statistically significant) in red blood cell cholinesterase activity in High dose rats. The High dose group showed increased creatinine levels (7%, P<0.05) compared to Controls. The statistical significances achieved by the Low and Intermediate dose group albumin-globulin levels (P<0.01 in both cases) are considered to be chance effects as the High dose group was the same as Controls and there was no notable effect on the other protein parameters.

Week 51: Sodium levels were decreased in all groups receiving test item with the Low and High dose groups achieving statistical significance (3%, P<0.01). All groups receiving test item showed lower phosphate levels than Controls; Low dose -15%, P<0.01; Intermediate dose -9%, P<0.05; High dose -12%, P<0.01, Calcium levels were also slightly reduced in the High dose group only (5%, P<0.01). The High dose group once more showed reduced (32%, P<0.01) red blood cell cholinesterase levels compared to Control.

Week 100 (Carcinogenicity Study Animals): All groups receiving test item showed lower plasma cholinesterase levels than Controls, but as the dose response was inverted and a high degree of individual variation was present, this was considered to be a chance effect. RBC and brain cholinesterase levels showed no intergroup differences.
The only other intergroup difference to show statistical significance was a reduction in High dose calcium levels (6%, P<0.01). Although there was evidence of a dose relationship and this parameter had shown a slight reduction at Week 51, the effect is not considered to be of clinical importance.

Females
Week 13: All groups receiving test item showed lower plasma cholinesterase levels than Controls. This achieved statistical significance in the Intermediate (23%, P<0.05) and High (38%, P<0.001) dose groups.

Week 26: On this occasion only the High dose group showed a reduction (35%, P<0.05) in plasma cholinesterase levels. Other instances of statistical significance are considered to be chance effects.

Week 51: The Intermediate and High dose groups showed reductions (29%, P<0.05 and 39%, P<0.01, respectively) in plasma cholinesterase levels. These groups also showed reductions (15%, P<0.01 in both cases) in blood urea nitrogen levels.

Week 100 (Carcinogenicity Study Animals): As at previous timepoints the High dose group showed lower plasma cholinesterase levels than Controls (14%, P<0.05). RBC and brain cholinesterase levels showed no intergroup differences.
In conclusion, changes in clinical chemistry parameters were not considered to be of toxicological relevance, because they were not observed throughout the treatment period and/or were observed without a dose-response relationship. Furthermore, changes were of low magnitude and are therefore considered to be of normal biological variation.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no notable intergroup differences in either sex.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The only notable intergroup differences that could be correlated with histopathological findings were associated with the stomachs of Intermediate dose males and High dose males and females, where the incidences of depressed and raised foci were increased.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Week 52 Sacrifice:

Stomach:
Acanthosis was present as a significant treatment related change in the High and Intermediate groups of both sexes although it was also present in 2 Control and 4 Low dose males. Hyperkeratosis, present in a number of groups was only significant in the Intermediate dose group males. Submucosal inflammation was a treatment related change in female rats of the High dose group. Other changes, that did not achieve significance, but were possibly related to treatment, by pathological association, were ulceration, submucosal oedema and inflammation of the keratin layer itself. With the exception of acanthosis, the gastric lesions in the Low dose males were seen as only single incidences and never were 2 lesions seen in the same animal. These findings were not seen in the Low dose females.
All other changes observed were not related to treatment and were those neoplastic and non-neoplastic changes that are commonplace in laboratory rats of this age and strain.

Premature Decedents: The premature decedent High dose male (in which the gastric section was examined) had the apparent treatment related lesions of acanthosis and keratin layer inflammation. There were no severe lesions in the other 2 premature decedents.


Week 104 Sacrifice:

There were a number of treatment related findings. The major effects were seen in the keratinized stomach and salivary glands of both sexes. In the keratinized stomach of the Intermediate and High dose groups of both sexes, there were increased incidences of submucosal inflammation, acanthosis, hyperkeratosis, and basal cell proliferation. Significant increases in the incidences of submucosal oedema were seen only in the male Intermediate and High dose groups. Inflammation of the keratin layer also affected the male High dose group. There were also increased incidences of ulceration in the male High dose group and in the female Intermediate and High dose groups. Lesions associated with ulceration were increased in the female Intermediate and High dose groups. Salivary gland lobular degeneration was increased in both the male and female Intermediate and High dose groups, but in addition, the male High dose group also showed increased incidences of fibroplasia.
Other findings that were increased in incidence in the male Intermediate and High dose groups included pulmonary foamy macrophage aggregates and thyroid colloid basophilia.
Other findings which were increased in incidence in females included compression of the brain thalamus in the Low and Intermediate dose groups, and internal hydrocephalus in the Intermediate dose group. The lack of these effects in the High dose groups eliminated these changes as being truly related to treatment and are consequently believed to result from chance incidences.
In the males there were 2 findings that were reduced with treatment; vacuolation of the lumbar spinal cord in the High dose group and basophilic cell foci of the liver in all dose groups which received test item.
Other changes which occurred with greater frequency in the male High dose group than in the Control group included cardiac chronic active myocarditis and pancreatic lobular degeneration. These changes were statistically significant only in the terminal kill animals, and ceased to be statistically significant when combined with decedents and those killed prematurely.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Week 52 Sacrifice:
There were occasional neoplasms present. The incidence of these indicated that there was not a treatment related change.

Week 104 Sacrifice: There were no neoplasms that showed a treatment related increase.
In females, the incidence of mammary fibroadenomas was increased in the Low dose group and the incidence of pituitary adenomas was increased in the Intermediate dose group. These increases are regarded as spurious. A consideration of the overall tumour incidence in the Control and High dose groups did not indicate a treatment related increase in either sex.

Other effects:

no effects observed

Description (incidence and severity):

Cholinesterase activity:
At termination, plasma cholinesterase activity was statistically significantly decreased in all treated males, which is considered as a result of high plasma cholinesterase activity in concurrent control. Reduced plasma cholinesterase activity was observed in females at High-Dose group and occasionally in Mid-dose group. Whereas RBC cholinesterase was reduced at only one single time point and thus, a dose-response relationship is lacking. Furthermoe, no difference in brain cholinesterase is activity was observed.

Details on results:

Analysis of formulated diets:
Generally, all diets were within acceptable limits of accuracy and homogeneity (+/- 15 %). Only occasional diets fell outwith this limit; analyses of these diets were repeated at subsequent mixes and proved to be satisfactory showing that there was no continuing problem with mixing.

Key result

Dose descriptor:

LOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Table 1. Two-year Combined Chronic/Carcinogenicity Study in Rats

Parameter

0 mg/kg bw/d

20 mg/kg bw/d

40 mg/kg bw/d

80 mg/kg bw/d

dose-response + / -

m

f

m

f

m

f

m

f

m

f

Number of animals in group

50

50

50

50

50

50

50

50

Mortality

22

17

20

21

24

10

14

14

-

-

clinical signs

ne

ne

ne

ne

ne

ne

ne

ne

-

-

body weight gain [% of control]

89

99

86

89

71

76

+

+

total food consumption

ne

ne

ne

ne

ne

ne

ne

ne

clinical chemistry

ne

ne

ne

ne

ne

ne

ne

ne

haematology

ne

ne

ne

ne

ne

ne

ne

ne

urinalysis

ne

ne

ne

ne

ne

ne

ne

ne

Liver

adjusted weight

interim kill

19.12

10.32

18.28

10.93

19.58

12.30***

20.60**

11.72**

+

+

terminal kill

18.67

14.72

20.41

12.93

20.11

14.67

18.43

13.46

-

-

gross pathology

interim kill

ne

ne

ne

ne

ne

ne

ne

ne

-

-

terminal kill

dark red foci

5

11

3

13

8

17

10

19

+

+

histopathology

interim kill

ne

ne

ne

ne

ne

ne

ne

ne

-

-

terminal kill

basophilic cell focus/ area

12/50^a)

24/50

2**/50

17/49

2**/50

22/50

3*/50

18/50

+

-

Submaxillary salivary glands

terminal kill

lobular degeneration

1/49

1/50

3/50

4/49

16***/50

20***/50

26***/49

26***/50

+

+

fibroplasia

0/49

0/50

1/50

0/49

1/50

2/50

6*/49

0/50

-

-

Thyroid 1terminal kill

colloid basophilia

0/49

1/50

0/22

2/21

5**/29

1/10

6*/49

2/50

+

-

Thyroid 2terminal kill

colloid basophilia

0/48

1/48

0/23

2/21

2/24

1/10

6*/48

2/49

+

-

Lungsterminal kill

foamy macrophage aggregates

3/50

3/50

8/49

5/50

14**/50

6/50

18***/50

9/50

+

-

Stomach

gross pathology

interim kill

erosions

0

0

0

0

1

0

1

4

-

+

terminal kill

mass

0

0

0

0

2

0

2

0

+

-

depressed foci

4

0

8

4

17

7

21

15

+

+

Stomach keratinsed/Forestomach

interim kill

submucosal oedema

0/15

0/15

1/15

0/15

1/14

2/15

0/14

2/15

-

-

submucosal inflammation

0/15

0/15

1/15

0/15

1/14

2/15

1/14

8/15

-

+

acanthosis

2/15

0/15

4/15

0/15

12/14

4/15

8/14

8/15

+

+

inflammation of keratin layer

0/15

0/15

1/15

0/15

1/14

2/15

1/14

3/15

-

+

hyperkeratosis

0/15

0/15

1/15

0/15

5/14

3/15

1/14

1/15

-

-

ulceration

0/15

0/15

0/15

0/15

1/14

0/15

0/14

3/15

-

-

basal cell proliferation

0/15

0/15

0/15

0/15

0/14

2/15

1/14

0/15

-

-

terminal kill

submucosal oedema

4/49

2/50

7/49

1/49

15**/48

7/50

19***/49

6/50

+

-

submucosal inflammation

5/49

1/50

9/49

3/49

26***/48

8*/50

28***/49

20***/50

+

+

acanthosis

13/49

8/50

13/49

7/49

37***/48

28***/50

43***/49

38***/50

+

+

inflammation of keratin layer

0/49

0/50

0/49

0/49

5*/48

5/50

10**/49

2/50

+

-

hyperkeratosis

1/49

0/50

5/49

3/49

24***/48

13***/50

29***/49

21***/50

+

+

ulceration

3/49

0/50

5/49

3/49

10*/48

6*/50

16**/49

12***/50

+

+

basal cell proliferation

4/49

3/50

7/49

1/49

28***/48

11*/50

45***/49

39***/50

+

+

lesions associated with ulcer

5/49

0/50

4/49

3/49

9/48

6*/50

13/49

17***/50

+

+

Significance from control: * p < 0.05; ** p < 0.01; *** p < 0.001

^a)             number of animals with findings/number of animals examined

Abbreviations:

ne           no effects

ni            not indicated

 

Conclusions:

Dosing Sprague-Dawley rats with test item via the diet for 104 weeks produced reductions in body weight gain at 20, 40 and 80 mg/kg bw/day in males and at 40 and 80 mg/kg bw/day in females. Both males and females receiving 40 or 80 mg/kg bw/day showed increased incidences of lesions in the keratinised stomach (submucosal oedema and inflammation) and submaxillary salivary glands (lobular degeneration).
There was no evidence of carcinogenic potential in either sex. A no observable effect level in terms of major structural effect can be set out at 20 mg/kg bw/day in both sexes.

Executive summary:

Groups of 50 male and 50 female Sprague-Dawley rats were offered diet ad libitum formulated with test item at concentrations calculated to give dose levels of 20, 40 and 80 mg/kg bw/day. A further group of 50 males and 50 females were offered untreated diet to act as Controls. During Weeks 53/54, 79 and 104 a blood smear was prepared via tailsnip from all surviving animals and a differential blood count performed on Control and High dose group animals. During Week 100 blood samples were taken from 10 males and 10 females from each group in order that a haematology and clinical chemistry screen could be carried out in order to put in context results from earlier time points on the concurrent toxicity study.

After 104 weeks continual dosing all surviving animals were killed by carbon dioxide asphyxiation followed by exsaguination and a detailed necropsy, with a selection of organs being placed in fixative. All organs taken were examined histopathologically from all Control and High dose animals, as well as premature decedents from the Low and Intermediate dose groups. In addition liver, kidneys, lungs, stomach and salivary glands were examined from all survivors in the Low and Intermediate dose groups.

Regarding mortality, there were no notable intergroup differences observed. There were no clinical signs thought to be attributable to dosing with the test item. There was a reduction in body weight gain in all male dose groups which receive test item ranging from slight in the Low dose group to marked in the High dose group. There was also a moderate reduction in body weight gain in the female Intermediate dose group and a marked reduction in the female High dose group. A slight reduction in food consumption in the male dose group receiving 80 mg/kg bw/day was found. There were no visual intergroup differences observed regarding water consumption.

There were no notable intergroup differences at any time in regards to differential blood counts nor in regards to organ weights in either sex. Intermediate dose males and High dose males and females showed increased incidences of depressed and raised foci in the stomach. Effects of treatment were present in the salivary glands and stomach of Intermediate and High dose groups in both sexes. In the submaxillary salivary gland lobular degeneration was evident in both groups, both sexes. In the keratinized stomach of both groups, in both sexes, notable intergroup differences were seen for submucosal oedema, submucosal inflammation, acanthosis, hyperkeratosis, inflammation in the keratinized layer, ulceration, basal cell proliferation and 'lesions associated with ulcer'.

Accroding to a subsequent pathological review and interpretation of selected thyroid and forestomach lesions, lesions reported are not thought to pose a potential carcinogenic risk to humans.
There were no lesions reported in the thyroid which were thought to be related to administration of the test item. The increased incidences of colloid basophilia of the thyroid reported in mid and high dose male rats represented several artifacts which happened to be reported more commonly in these treatment groups.
Administration of the test item was associated with non-neoplasitc proliferative and inflammatory lesions of the forestomach at 40 and 80 mg/kg bw/d in both male and female rats. These lesions are not thought to pose a potential carcinogenic risk to humans.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

GLP and guideline study

System:

hepatobiliary

Organ:

liver

thyroid gland

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on chronic toxicity, the test item is not considered to be classified as carcinogen according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.

Additional information

Carcinogenicity study in mice

Groups of 50 male and 50 female CD-I mice were dosed with the test item via the diet at concentrations calculated to achieve dose levels of 20 (Low dose), 50 (Intermediate dose) and 150 (High dose) mg/kg bw/d. A further group of 50 males and 50 females received untreated diet to act as Controls. During Weeks 52 and 78 blood samples were taken from all animals via a tailsnip and a differential blood smear prepared. A blood sample was also taken via the orbital sinus during Week 78 which was used for clinical chemistry. After 78 weeks of dosing all remaining animals were killed by carbon dioxide asphyxiation followed by exsanguination, followed by a gross necropsy. Histopathological evaluation was carried out.
There were no notable intergroup differences in either sex regarding mortality. There were no notable intergroup differences at clinical signs parameters. There was a moderate reduction in body weight gain in the female High dose group and a marked reduction in the male High dose group. However, in regards to food consumption, there were no notable intergroup differences. There were no notable intergroup differences in neither differential blood count nor clinical chemistry parameters.
AT gross pathological examination there was an increased incidence of thyroid enlargement in the male High dose group. Histopathological investigation revealed an increased incidence of hepatic adenoma in the male High dose group. There were unusual changes in thyroids which were thought to be atrophic and were not thought to be proliferative. They comprised atrophic follicular vacuolation, generalised follicular enlargement, follicle coalescence and chronic follicular inflammation seen singly or in some combination in all dose groups.

Subsequently, an independent panel review of the livers by another working group was performed. Evaluation of liver sections resulted in the conclusion that the test item given to male and female CD-I mice in the diet at levels of 20, 50, or 150 mg/kg bw/d for 78 weeks did not result in any carcinogenic effect. The incidence of hepatocellular adenomas in high dose males was increased over controls. However, this increase was not considered to be biologically relevant as there was no increase in the incidence of hepatocellular carcinomas, foci of cellular alteration, or combined benign and malignant hepatocellular tumors. There was no increase in the incidence of hepatocellular tumors or foci of cellular alteration in female mice from any treatment group.

Carcinogenicity study in rats

Groups of 50 male and 50 female Sprague-Dawley rats were offered diet ad libitum formulated with test item at concentrations calculated to give dose levels of 20, 40 and 80 mg/kg bw/day. A further group of 50 males and 50 females were offered untreated diet to act as Controls. During Weeks 53/54, 79 and 104 a blood smear was prepared via tailsnip from all surviving animals and a differential blood count performed on Control and High dose group animals. During Week 100 blood samples were taken from 10 males and 10 females from each group in order that a haematology and clinical chemistry screen could be carried out in order to put in context results from earlier time points on the concurrent toxicity study.

After 104 weeks continual dosing all surviving animals were killed by carbon dioxide asphyxiation followed by exsaguination and a detailed necropsy, with a selection of organs being placed in fixative. All organs taken were examined histopathologically from all Control and High dose animals, as well as premature decedents from the Low and Intermediate dose groups. In addition liver, kidneys, lungs, stomach and salivary glands were examined from all survivors in the Low and Intermediate dose groups.

Regarding mortality, there were no notable intergroup differences observed. There were no clinical signs thought to be attributable to dosing with the test item. There was a reduction in body weight gain in all male dose groups which receive test item ranging from slight in the Low dose group to marked in the High dose group. There was also a moderate reduction in body weight gain in the female Intermediate dose group and a marked reduction in the female High dose group. A slight reduction in food consumption in the male dose group receiving 80 mg/kg bw/day was found. There were no visual intergroup differences observed regarding water consumption.

There were no notable intergroup differences at any time in regards to differential blood counts nor in regards to organ weights in either sex. Intermediate dose males and High dose males and females showed increased incidences of depressed and raised foci in the stomach. Effects of treatment were present in the salivary glands and stomach of Intermediate and High dose groups in both sexes. In the submaxillary salivary gland lobular degeneration was evident in both groups, both sexes. In the keratinized stomach of both groups, in both sexes, notable intergroup differences were seen for submucosal oedema, submucosal inflammation, acanthosis, hyperkeratosis, inflammation in the keratinized layer, ulceration, basal cell proliferation and 'lesions associated with ulcer'.

Accroding to a subsequent pathological review and interpretation of selected thyroid and forestomach lesions, lesions reported are not thought to pose a potential carcinogenic risk to humans.
There were no lesions reported in the thyroid which were thought to be related to administration of the test item. The increased incidences of colloid basophilia of the thyroid reported in mid and high dose male rats represented several artifacts which happened to be reported more commonly in these treatment groups.
Administration of the test item was associated with non-neoplasitc proliferative and inflammatory lesions of the forestomach at 40 and 80 mg/kg bw/d in both male and female rats. These lesions are not thought to pose a potential carcinogenic risk to humans.