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Description of key information

30-day and 90-day rat oral (gavage) studies are available.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 December 2012 to 7 December 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test guideline and GLP compliant study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: 179-229g males and 123-172g females
- Fasting period before study: No
- Housing: Housed in groups of two or three by sex in suspended polycarbonate cages
- Diet (e.g. ad libitum): rat & mouse modified No. 1 diet SQC expanded available ad libitum
- Water (e.g. ad libitum): ad libitum access to cage-mounted water bottles
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 44-55
- Air changes (per hr): 10/hr
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 4 December 2012 To: 13 March 2013
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test formulations were prepared weekly and stored at ambient laboratory temperature (<30°C)

VEHICLE
- Concentration in vehicle: 0, 10, 40 and 160 mg/mL (adjusted concentration to take account of test substance purity - 0, 10.8, 43.3, 173.3 mg/mL)
- Amount of vehicle (if gavage): 5 ml/kg bw/d
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were analysed for achieved concentration and homogeneity in Weeks 1, 2,6 and 12 using gas chromatography with flame ionisation detection. Stability analyses were also completed over a concentration range of 0.1 to 200 mg/ml to determine stability for 8 days.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 50, 200, 800 mg/kg bw/d
Basis:
other: nominal concentration
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of a 14 day preliminary study
- Rationale for animal assignment (if not random): randomised
Positive control:
Not applicable to this study type
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily observations

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly from pre-dose period and throughout study to check appaerane. movement and behavioural responses, skin , hair and eye condition, mucus membranes, respiration and excreta

BODY WEIGHT: Yes
- Time schedule for examinations: weekly intervals from Day 0 (Immediately prior to dosing)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined : Yes recorded at weekly intervals

WATER CONSUMPTION : Yes
- Time schedule for examinations: Monitored visually throughout the study

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:pre-dose and week 13
- Dose groups that were examined: Eyes of all animals examined prior to initial dose administration and control and high dose animals re-examined in week 13. indirect opthalmoscpe and mydriatic agent used for anterior, lenticular and fundus examinations

HAEMATOLOGY: Yes
- Time schedule for collection of blood: all animals during week 13/14 - day 92/93.
- Anaesthetic used for blood collection: Yes isoflurane for orbital sinus sample collection
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table 1 and 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: all animals during week 13/14 - day 92/93.
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked in table 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:all animals during week 13 - day 87/88.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes 4 hr colllection period with food and water deprivation
- Parameters checked in table 4 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: pre-dose and week 13
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity / grip strength / motor activity - see further information for additional detail of the FOB

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy, tissue collection and organ weights were recorded for all animals in all groups at scheduled termination at day 92/93

HISTOPATHOLOGY: Yes
Full tissue list examined histopathologically for control and high dose groups; gross lesions, liver and sternum also examined for the low and intermediate groups.
Other examinations:
A functional observational battery of tests was included once prior to initial dosing and again during week 13.
The standard FOB parameters were checked including cageside observations, removal from cage assessments, handling, ocular condition, body temperatures, haircoat condition, standardised arena observations over 2 minutes (latency, mobility grooming, rearing, excretion, posture, gait abnormalities and stereotypy), functional tests - auditory startle response, grip strength, touch reaction, pain perception, landing/foot splay and motor activities.
Statistics:
Body weight, food consumption, selected functional observation battery and motor activity data, haematology, coagulation, clinical chemistry and selected urinalysis data was analysed for homogeneity of variance using the ‘F Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test i.e., pairwise comparisons were made only if the overall F test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
In circumstances where it was not possible to perform the F Max test due to zero standard deviation in at least one group, the non-parametric ANOVA results were reported.
Organ weights were analysed using ANOVA as above and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. In addition, organ weights as a percentage of terminal body weight were analysed using ANOVA as above.

In circumstances where the variances in the ANCOVA remained heterogeneous following log or square root transformations, the data was subjected to a rank transformation prior to analysis. Where it was not possible to perform the F-Max test due to the small sample size (i.e., less than 3 animals in any group), the untransformed parametric ANCOVA results were reported.

In the ANOVA and ANCOVA summary tables, the results of the analysis were reported indicating the level of statistical significance (p<0.05, p<0.01 and p<0.001) of each pairwise comparison.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs of toxicity (piloerection, subdued behaviour) in both sexes at 800 mg/kg bw/d
Mortality:
mortality observed, treatment-related
Description (incidence):
Signs of toxicity (piloerection, subdued behaviour) in both sexes at 800 mg/kg bw/d
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significanty reduced in males at 200 and 800 mg/kg bw/d; not considered to be of toxicological significance at 200 mg/kg bw/d
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Altered red blood cell parameters in males at 800 mg/kg bw/d
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Increased volume and low pH at 800 mg/kg bw/d
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased liver weights at 800 mg/kg bw/d and in males at 200 mg/kg bw/d
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Isolated liver centrilobular hypertrophy in males at 200 mg/kg bw/d. Liver and bone marrow pathology in males and females at 800 mg/kg bw/d
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths.

All high dose males and females showed a behavioural change in which the animal ploughs its nose through substrate and bedding immediately after dosing. The effect was observed first on Day 10 or 14 and persisted through the dosing period. Excess salivation was observed in all treated animals showing a degree of dose-relationship. Findings are attributable to gustatory perception and are not considered to be of toxicological significance. These signs were also observed transiently in the control group.

Sporadic incidents of reduced activity and subdued behaviour were observed in males and females treated at 800 mg/kg bw/day, and piloerection and abnormal respiration were also noted in a few rats at this dose level.

No abnormal clinical changes were apparent for the low and intermediate dose groups.

BODY WEIGHT AND WEIGHT GAIN

Male rats dosed at 200 or 800 mg/kg bw/day showed significant, dose-related lower bodyweight gains over the dosing period. No effect on female weights. The magnitude of change in males at 200 mg/kg bw/d was low and is not considered to be ot toxicological significance.

FOOD CONSUMPTION
No treatment-related biologically significant change in food consumption.

WATER CONSUMPTION
No observable effects on water consumption

OPHTHALMOSCOPIC EXAMINATION
No ophthalmic changes considered attributable to treatment

HAEMATOLOGY
Significant changes were apparent in some haematological parameters. In the high dose group, 800 mg/kg bw/d, lower red cell values were recorded affecting haemoglobin, mean cell haemoglobin and mean cell haemoglobin concentration and lower mean cell volume. Lower reticulocyte values were also recorded for males dosed at 800 mg/kg bw/day. No other haematology changes were considered biologically significant.

CLINICAL CHEMISTRY
There were no clinical chemistry findings that were related to treatment.

URINALYSIS
The urinary volume was higher than control for both males and females dosed at 800 mg/kg bw/d and the urinary pH was lower in this group also. But in the absence of any histopathological or clinical chemistry effects associated with renal change, the significance of urinary effects in the high dose group was unclear.


NEUROBEHAVIOUR
The functional observation battery results for the detailed clinical observations showed no treatment-related differences at any dose level.
There were no treatment-related differences in any of the quantitative functional observation parameters.
The motor activity assessments showed no notable inter-group differences although the number of overall movements of males and females dosed at 200 or 800 mg/kg bw/d were lower than controls. However comparison of pre-dose motor activity with week 13 activity gave no indication of any treatment-related effects.


ORGAN WEIGHTS
No significant effects were noted on any of the measured organ weights although a slight non-significant increase in liver weight was apparent in the high dose group.


GROSS PATHOLOGY
No treatment related macroscopic abnormalities were apparent.


HISTOPATHOLOGY: NON-NEOPLASTIC
Centrilobular hypertrophy was recorded in males that received ≥200 mg/kg bw/d and females that received 800 mg/kg bw/d while biliary pigment, peribiliary inflammatory cell infiltration, bile duct hyperplasia, and centrilobular inflammation were also observed in males that received 800 mg/kg bw/d Other changes included decreased haemopoiesis in the sternum of a single male that received 800 mg/kg bw/d.
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Clinical signs, liver weight and liver histopathology in both sexes at 800 mg/kg bw/d
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Marginal decrease in weight gain and adpative liver changes at 200 mg/kg bw/d
Dose descriptor:
LOAEL
Effect level:
800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Clinical signs, liver weight and liver histopathology in both sexe
Critical effects observed:
not specified

Summary of findings

 

M

F

Dose level (mg/kg bw/d)

0

50

200

800

0

50

200

800

Ploughing (#)

2

1

6

10

2

-

7

10

Salivation

3

10

10

10

3

9

10

10

Subdued behaviour

-

1

-

7

-

-

1

3

Abnormal breathing

-

-

-

2

-

-

-

2

Terminal bodyweight (g)

430

411

407

399

244

247

227*

248

Weight gain (g)

227

207

197*

193*

96

100

91

95

Hb (g/dL)

15.8

15.4

15.6

15.1**

14.6

14.4

14.5

14.0

MCH (pg)

17.7

17.4

17.3

16.8**

18.2

18.3

18.2

17.9

MCHC (g/dL)

33.8

33.7

33.6

33.0**

34.0

34.1

33.9

33.9

Liver weight (g)

14.73

14.19

14.97

16.49

8.41

8.57

7.94

10.50***

Liver weight (relative)

3.440

3.462

3.688*

4.166***

3.441

3.481

3.528

4.308***

Liver centrilobular hypertrophy

-

-

3

6

-

-

-

9

Biliary pigment

-

-

-

2

-

-

-

-

Liver: inflammatory infiltration

-

-

-

3

-

-

-

-

Bile duct hyperplasia

-

-

-

1

-

-

-

-

Liver centrilobular hyperplasia

-

-

-

2

-

-

-

-

Sternum reduced haematopoiesis

-

-

-

1

-

-

-

-

*significantly different to controls ([<0.05); **p<0.01; *** p<0.001

Conclusions:
A NOAEL of 200 mg/kg bw/d is derived for this study based on clinical signs, increased liver weights and associated pathology at the highest dose level of 800 mg/kg bw/d. A minimal reduction in weght gain and adpative liver changes seen in males at 200 mg/kg bw/d are not considered to be adverse.
Executive summary:

TMPDE was administered orally by gavage to groups of ten males and ten female rats over a 90-day treatment period. The study was conducted in compliance with the OECD Test Guideline No. 408 and EC Directive 2001/59, Method B.26. TMPDE was dispersed in corn oil and dose concentrations were adjusted for the test substance purity. Parameters including viability, clinical signs, bodyweights, food consumption, water consumption, ophthalmoscopy, a functional observation battery with motor activity assessments; blood samples were collected during week 13 for haematology, coagulation and blood chemistry investigations; urine samples were also collected during week 13. Necropsy following completion of the dosing period on Day 91 included a full macroscopic examination and weighing of selected organs. Control group and high dose tissues were retained for histopathological examinations. Liver and sternum tissues were also examined from the low and intermediate dose groups.

There were no mortalities during the treatment period. Clinical observations of reduced activity or subdued behaviour for the majority of males and females dosed at 800 mg/kg bw/d were accompanied by piloerection and abnormal respiration. Ploughing, where the animal ploughs its nose through bedding and cage substrate, was observed in the high dose animals. Excessive salivation was noted in all treated animals. Males treated at 200 or 800 mg/kg bw/d showed significantly lower bodyweight gains in comparison with the controls; however the effects at 200 mg/kg bw/d are minimal and not considered to be of toxicological significance. No effect on food consumption was apparent. There were no ophthalmoscopic changes apparent. Lower haemoglobin, lower mean cell volume, mean cell haemoglobin and mean cell haemoglobin concentration were observed in the high dose males and lower reticulocytes were also apparent in the males dosed at 800 mg/kg bw/d. Elevated liver weights were apparent for the males dosed at 800 or 200 mg/kg bw/d. Other organ weight changes included higher female kidney weights in the high dose group; and lower male spleen weights for the high dose group. The microscopic examinations confirmed centrilobular hypertrophy in the males treated at 200 or 800 mg/kg bw/d and the high dose females. Biliary pigments, peribiliar inflammatory cell infiltration , bile duct hyperplasia and centrilobular inflammatory responses were evident for the high dose male rats. One high dose male also had decreased haemopoeisis in the sternum.

The NOAEL was 200 mg/kg bw/d for both male and female rats based on observations of liver weight change and possible adaptive effects on other liver parameters, in conjunction with bodyweight changes and adverse clinical obsservations at the highest dose level. . The minimal reduction in weght gain and adpative liver changes seen in males at 200 mg/kg bw/d are not considered to be adverse.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A modern and guideline-compliant 90-day rat study is supported by an older 30-day rat study. The findings of both of these studies are consistent.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The toxicity of trimethylolpropane diallyl ether (TMPDE) was evaluated in a 30-day oral toxicity study, according to OECD method 407. Groups of 5 male and 5 female Wistar rats were administered doses of 0, 50, 200 or 800 mg/kg daily for 30 days by gavage. There were no clinical signs of toxicity observed throughout the study. Mortality, body weight gain and feed and water intake were unaffected by treatment. Biliary hyperplasia and pericholangitis/cholangitis was diagnosed in the livers of 800 mg/kg bw/d males. These animals also demonstrated decreased cholesterol and bilirubin concentrations in peripheral blood, and slightly increased liver weights. There was no effect on the liver in females. No other effects of treatment were observed, therefore the 30 day NOAEL for male rats was 200 mg/kg bw/d, whilst the 30 day NOAEL for female rats was 800 mg/kg bw/d.

In a modern, GLP- and guideline-compliant 90-day rat study, clinical observations of reduced activity or subdued behaviour for the majority of males and females dosed at the highest dose level of 800 mg/kg bw/day were accompanied by piloerection and abnormal respiration. Ploughing, where the animal ploughs its nose through bedding and cage substrate, was observed in the high dose animals. Excessive salivation was noted in all treated animals. Males treated at 200 or 800 mg/kg bw/d showed significantly lower bodyweight gains in comparison with the controls; however the effects at 200 mg/kg bw/d are minimal and not considered to be of toxicological significance. No effect on food consumption was apparent. There were no ophthalmoscopic changes apparent. Lower haemoglobin, lower mean cell volume, mean cell haemoglobin and mean cell haemoglobin concentration were observed in the high dose males and lower reticulocytes were also apparent in the males dosed at 800 mg/kg bw/d. Elevated liver weights were apparent for the males dosed at 800 or 200 mg/kg bw/d. Other organ weight changes included higher female kidney weights in the high dose group; and lower male spleen weights for the high dose group. The microscopic examinations confirmed centrilobular hypertrophy in the males treated at 200 or 800 mg/kg bw/d and the high dose females. Biliary pigments, peribiliar inflammatory cell infiltration , bile duct hyperplasia and centrilobular inflammatory responses were evident for the high dose male rats. One high dose male also had decreased haemopoeisis in the sternum.

The NOAEL was 200 mg/kg bw/d for both male and female rats based on observations of liver weight change and possible adaptive effects on other liver parameters, in conjunction with bodyweight changes and adverse clinical observations at the highest dose level. . The minimal reduction in weght gain and adapative liver changes seen in males at 200 mg/kg bw/d are not considered to be adverse.

Waivers are proposed for testing by the dermal and inhalation routes. Adequate data are available for the oral route. The substance is a non-volatile liquid and significant inhalation exposure is not predicted.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Modern, guideline-compliant study is of the longest duration and in the preferred species

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

The available data indicate that TMPDE is of low toxicity and is not classified for repeated dose effects.