4-hydroxy-2,2,6,6-tetramethylpiperidinoxyl

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Based on the findings in this study the female No Observed Adverse Effect Level (NOAEL) is considered to be 125 mg/kg/day, and foetal NOAEL is considered to be 400 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 January 2017 - 27 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Current requirements of the Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 114
- Purity: 98.4%
- Expiration date of the lot/batch: Restest 12 October 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Sealed container, refrigerated (2 to 8 °C), in the dark
- Storage condition of formulated test material: Sealed container, room temperature (15 to 25 °C), in the dark
- Stability under test conditions: Stable for 48 days

VEHICLE
- Vehicle: Polyethylene glycol 400 (PEG 400)
- Supplier: Merck

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sex: female
- Age at mating: 8 to 10 weeks old at mating
- Weight at study initiation (gestation day 3): 171.5 and 252.0 g
- Fasting period before study: No
- Housing: Animals were housed individually. Bedding, Aspen wood chips, was changed weekly. Animals were provided with wooden Aspen chew blocks and nesting materials as forms of environmental enrichment.
- Diet: ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 9-27 January 2017

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Preparation: Formulations were stirred continuously before dosing commenced (excluding those for the control article [vehicle]) and throughout dosing.
- Rate of preparation of dose (frequency): Formulations were prepared daily.
- Storage temperature of prepared doses: Formulations were stored at room temperature (15 to 25 °C) in a sealed container, protected from the light.

DOSE VOLUME: 10 mL/kg bw

VEHICLE
- Concentration in vehicle: 0, 4, 12.5 and 40 mg/mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulations of the test material, prepared at 4 and 40 mg/mL (in Polyethylene Glycol 400 (PEG400)), were analysed to determine homogeneity and stability. Formulations were determined to be homogeneous and stable for 50 days when stored at 2 to 8 °C.
Formulations prepared for use on the first and last day of dosing were analysed to determine the achieved concentration. The mean % target range for the preparation of formulations was 90 to 110 % of nominal. Results were within this range. Test material was not detected in Group 1 control samples. The analytical procedure was validated and all acceptance criteria for the method were met.

Triplicate samples of 3 mL were removed from the top, middle and bottom of the test material formulations per Group and control formulations and were analysed by gas chromatography (GC) to determine concentration. Samples were diluted in methanol and analysed against a standard.

GC SYSTEM PARAMETERS
Column (analytical): DB1, 30 m x 0.32 mm id, 0.25 µm df
Column (guard): Deactivated, 1 m x 0.32 mm id
Inlet liner: Single taper, deactivated split liner with glass wool, e.g. Restek Sky® 23309
Carrier gas: Helium @ 2.5 mL/minute constant flow
Oven temperature program: 80 °C hold for 10 minutes; 10 °C/minute t 150 °C, hold for 2 minutes; 25 °C/minute to 300 °C, hold for 10 minutes
Injection volume: 1 µL
Run time: 25 minutes
Inlet temperature: 300 °C
Split ratio: 10:1
FID temperature: 350 °C
Detector gas flow rates: hydrogen 40 mL/minute; Air: 350 mL/minute; Makeup (nitrogen) 35 mL/minute
Injector wash solvent: Methanol (3 washes pre and post injection). 3 sample washes and 6 sample pumps prior to injection

Details on mating procedure:

Mating was carried out at the supplier's laboratory. Animals were delivered to testing facility on gestation day 3.
- Impregnation procedure: cohoused
- Length of cohabitation: 1 night
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

Daily from gestation day 6 - 20

Frequency of treatment:

Daily

Duration of test:

Females were maintained to gestation day 21

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

40 mg/kg bw/day

Dose / conc.:

125 mg/kg bw/day

Dose / conc.:

400 mg/kg bw/day

No. of animals per sex per dose:

22 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high dose level of 400 mg/kg/day was selected. Maternal toxicity was expected on the basis of some increased organ weights and haematological and splenic effects (secondary to the haematological effects) and microscopic evaluation of the kidney (considered part of the process producing relative weight increases) noted at 200 and 1000 mg/kg bw/day in a 28 day rat gavage study (Hita Research Laboratories, Japan Study Number D-3213). These parameters were not evaluated in this study.
The intermediate dose of 125 mg/kg/day was selected as it approximates the top dose/root 10. It was expected to be a maternal no observed effect level (NOEL) on the basis of a 28 day gavage study (Hita Research Laboratories, Japan Study Number D 3213).
The low dose of 40 mg/kg/day was selected as it approximates the mid dose/root 10 and was included in the study as a potential NOEL if effects were observed at the intermediate-dose level.
- Rationale for animal assignment (if not random): Animals were assigned to dose groups based on their body weights at gestation day 0.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from the start of dosing until necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on GD 3, 6, 9, 12, 15, 17, 20, and 21.
Carcass Weight = Terminal body weight - uterus weight
Total Weight Change = Day 21 body weight - Day 3 body weight

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Determined daily from GD 3; calculated as g/animal/day.

HAEMATOLOGY: Yes
- Time schedule for examinations: Terminal blood samples for haematology (1 x 0.5 mL [EDTA], nominal) and coagulation (1 x 0.5 mL [trisodium citrate], nominal) were withdrawn from the abdominal aorta immediately prior to exsanguination from 11 animals per group.
- Parameters examined: Haemoglobin concentration, red blood cell count, packed cell volume, reticulocyte count, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, red cell distribution width, haemoglobin distribution width, total and differential white cell count, platelet count, platelet crit, mean platelet volume, platelet distribution width, prothrombin time, activated partial thromboplastin time and fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for examinations: Terminal blood samples for clinical chemistry (1 x 0.6 mL [lithium heparin] were withdrawn from the abdominal aorta immediately prior to exsanguination from 11 animals per group.
- Parameters examined: Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, sodium, potassium, calcium, inorganic phosphate, chloride, total protein, albumin, globulin, albumin:globulin ratio, total cholesterol, glucose, urea, total bilirubin and creatinine.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Food was not removed prior to scheduled necropsies. Females were sacrificed on GD 21 by cervical dislocation following isoflurane anaesthesia; death was confirmed by exsanguination. For females subjected to clinical pathology (haematology and clinical chemistry) following collection of the terminal blood samples animals were terminally sacrificed (under anaesthesia) by exsanguination.
- Examinations: Animals were weighed before necropsy. Animals were examined macroscopically. All lesions were recorded and retained in the relevant fixative. The ovaries and uteri were removed and examined. The uterus of any apparently non-pregnant female was immersed in a 10 % ammonium sulphide solution to reveal any evidence of implantation. For females subjected to clinical pathology. The spleen was dissected, freed from fat and other contiguous tissue, and weighed before fixation in 10 % neutral-buffered formalin.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Terminal body weight (recorded for adjusted gravid uterus weight calculations only and have not been reported). Early intrauterine deaths were classified as those which showed decidual or placental tissue only. Late intrauterine deaths showed embryonic or foetal tissue in addition to placental tissue. Dead foetuses were classified as those which appeared to have died shortly before necropsy. Implantations and foetuses were allocated numbers relating to their position in utero.

Fetal examinations:

- External examinations: Yes: all per litter
Approximately one half of the foetuses in each litter were dissected, and the viscera were examined. They were then eviscerated, and the carcasses were cleared in potassium hydroxide. The skeletons were stained with Alizarin Red S, retained in glycerol/propylene glycol, and examined for skeletal abnormalities.
The remaining foetuses were fixed in Bouin's fluid, and the viscera examined. The foetuses fixed for visceral examination were retained in the relevant fixative.
Fetal abnormalities were classified as malformations (rare and/or potentially lethal defects) and variations (commonly occurring non-lethal abnormalities).

Statistics:

Body weight, body weight gains, food consumption, clinical pathology parameters, absolute organ weights and organ:terminal body weight ratios were analysed using analysis of variance (ANOVA).
The number of corpora lutea, implantation sites, early and late resorptions, live foetuses, and % pre- and post-implantation loss were analysed using the Kruskal Wallis and Wilcoxon rank sum test.
Mean foetal weight (male, female, and combined) was analysed using analysis of covariance (ANCOVA), with litter size (live and dead foetuses) as the covariate. The number of corpora lutea, implantation sites, early and late resorptions, dead foetuses, live foetuses, percent pre- and post-implantation loss, and percent male foetuses were analysed using the Kruskal-Wallis and Wilcoxon rank sum test.
Gravid uterine weights and corrected body weights (carcass weights) were analysed using analysis of variance (ANOVA).
The percentage of foetuses affected (mean litter percent) was analysed using the Kruskal-Wallis and Wilcoxon rank sum test.
Proportion of litters affected was analysed using a one-sided upper tail Fisher’s exact test.

Indices:

% Pre-Implantation Loss = ((Number of corpora lutea - number of implantations) / Number of corpora lutea) x 100
% Post-Implantation Loss = ((Number of implantations - number of live embryos) / Number of implantations) x 100
% Male Foetuses = (Number of male foetuses / Number of foetuses of determined sex) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Immediately following administration, incidences of mouth rubbing, salivation, and paddling were observed for animals of all groups. These observations are noted in animals with this route of administration and are considered to be due to unpalatable vehicle and/or test articles and is not adverse.
Throughout the observation period, incidences of red/pink/orange stained fur, thinning fur, and minimal sores/lesions were noted in animals in all dose groups including control. One vehicle control female was also noted to have pale teeth. These are common findings often observed in animals of this age and strain and were considered not related to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Decreases in haemoglogin, red blood cell count and packed cell volume of 4-5 % were observed in animals administered 400 mg/kg/day, and were accompanied by increases in absolute numbers and the proportion of reticulocytes (39 and 45 % increases, respectively). Of these, the decreased haemoglobin concentration and increased reticulocyte count attained statistical significance.
All other changes were considered within normal background levels.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant increases in aspartate and alanine aminotransferase activities (60 and 33 % increases, respectively) were noted in animals in the 400 mg/kg bw/day group.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase in mean relative spleen weight of 23 % was noted for top-dose females. Increases in relative spleen weight at the mid and low doses were ca. 10 % or less and not dose related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The gastrointestinal tract finding of abnormal dark/red or red contents was noted in some females that littered early. This was likely placental material. This is an anticipated finding as the consumption of placental material following parturition is an anticipated behaviour in female rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

1, 3, 1 or 2 females administered 0, 40, 125 or 400 mg/kg/day were found to be non-pregnant and one female administered 400 mg/kg/day was found to have no viable foetuses. Pregnancy rates were reasonably consistent between the groups and within background data ranges. These rates of non-pregnancy were, therefore, considered incidental.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 125 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical biochemistry

haematology

organ weights and organ / body weight ratios

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no skeletal or visceral foetal malformations or variants at any dose level which were considered related to treatment.
The variant fixed visceral finding of kidney dilation was noted in litters from all groups including controls, but only the incidence for litters of females administered 125 mg/kg/day attained statistical significance. The incidences noted were outside the background data ranges for all treated groups; however, no dose-relationship occurred and during the fresh visceral exam, there was no concurrent increased incidence of the same finding apparent at any dose level. Considering the high background incidence in all groups following the fixed visceral exam, including controls, these changes likely represent an enhancement of a normal, underlying physiological processes and not a direct toxicological effect of treatment. These incidences are therefore considered of no toxicological significance, if not incidental.
Incidences of thymic remnant attained statistical significance in animals administered 400 mg/kg/day. However, these variant incidences are within the background range for this laboratory and therefore reflect normal biological variation and no effect of treatment.
Although a small number of other foetal malformations or variants were identified, none attained statistical significance. These malformations were disparate from other organ systems or remained either within the background data range or the incidence was similar to or less than those of controls. Furthermore, the majority of skeletal and visceral malformations seen in the high dose were related to just one multiply malformed foetus (Litter 86 foetus L5 had situs invertus, branched and fused ribs, absent or small cervical arch and centrum and also hemicentric thoracic centrum) and therefore, sporadic in nature and not related to the test item.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1: Summary of Reproductive Performance

	Dose Group (mg/kg)
	0	40	125	400
Total No. of females	22	22	22	22
Pregnant females	21	19	21	20
Non-pregnant females	1	3	1	2
Pregnant with total litter loss (NVF)	0	0	0	1
Females with live foetuses	21	19	21	19

NVF = No viable foetuses

 

Table 2: Summary of Caesarean Section Data

		Dose Group (mg/kg)
		0	40	125	400
Excluding female with no viable foetuses
No. pregnant at caesarean section	N	21	19	21	19
Corpora lutea	N Mean SD	21 12 1.9	19 12 2.0	21 12 1.8	19 12 1.5
Implantation sites	N Mean SD	21 11 1.7	19 11 1.4	21 11 2.4	19 11 2.4
Pre-implantation loss	N Mean SD	21 1 1.2	19 1 2.5	20* 1 2.0	19 1 1.6
Pre-implantation loss (%)	N Mean SD	21 9.9 9.56	19 8.2 13.49	20* 9.9 17.59	19 8.6 15.03
Early resorptions	N Mean SD	21 1 0.9	19 1 0.8	21 1 1.1	19 1 1.0
Late resorptions	N Mean SD	21 0 0.0	19 0 0.2	21 0 0.2	19 0 0.0
Total resorptions	N Mean SD	21 1 0.9	19 1 0.9	21 1 1.2	19 1 1.0
Dead foetuses	N Mean SD	21 0 0.0	19 0 0.0	21 0 0.7	19 0 0.0
Post-implantation loss	N Mean SD	21 1 0.9	19 1 0.9	21 1 1.3	19 1 1.0
Post-implantation loss (%)	N Mean SD	21 8.1 9.27	19 7.9 8.95	21 8.0 12.64	19 4.9 9.39
Live foetuses	N Mean SD	21 10 2.1	19 10 1.6	21 10 2.5	19 10 2.5
Gravid uterine weight (g)	N Mean SD	21 68.9 12.60	19 70.5 10.70	21 67.9 16.20	19 69.0 16.13
Corrected body weight (carcass weight; g)	N Mean SD	21 243.8 22.73	19 252.1 16.00	21 247.3 17.42	19 246.0 19.47
Corrected weight change days 3 to 21 (g)	N Mean SD	21 49.0 8.85	19 51.1 9.43	21 51.3 8.72	19 50.7 11.38
Total weight change days to 21 (g)	N Mean SD	21 117.9 15.59	19 121.6 12.54	21 119.2 16.97	19 119.7 19.70
No. of females with live foetuses	N	21	19	21	19
Mean number of male foetuses per litter	N Mean SD	21 5 1.6	19 5 1.8	21 5 1.6	19 5 1.9
Mean number of female foetuses per litter	N Mean SD	21 5 2.1	19 5 1.5	21 5 1.9	19 5 1.7
% Male foetuses	N Mean SD	21 48 16.4	19 49 14.1	21 47 11.4	19 51 13.0
Mean foetal weight (g)	N Mean SD	21 5.31 0.465	19 5.37 0.264	21 5.29 0.381	19 5.18 0.399
Mean weight male foetuses (g)	N Mean Adj. mean SD	21 5.46 5.46 0.504	19 5.54 5.53 0.279	21 5.40 5.39 0.344	19 5.32 5.34 0.425
Mean weight female foetuses (g)	N Mean Adj. mean SD	21 5.13 5.13 0.424	19 5.22 5.22 0.274	21 5.19 5.18 0.465	19 5.01 5.03 0.412
Including female with no viable foetuses
No. pregnant at caesarean section	N	21	19	21	20
Corpora lutea	N Mean SD	21 12 1.9	19 12 2.0	21 12 1.8	20 11 2.6
Implantation sites	N Mean SD	21 11 1.7	19 11 1.4	21 11 2.4	20 10 3.0
Pre-implantation loss	N Mean SD	21 1 1.2	19 1 2.5	20* 1 2.0	20 1 1.6
Pre-implantation loss (%)	N Mean SD	21 9.9 9.56	19 8.2 13.49	20* 9.9 17.59	20 8.2 14.76
Early resorptions	N Mean SD	21 1 0.9	19 1 0.8	21 1 1.1	20 1 1.0
Late resorptions	N Mean SD	21 0 0.0	19 0 0.2	21 0 0.2	20 0 0.0
Total resorptions	N Mean SD	21 1 0.9	19 1 0.9	21 1 1.2	20 1 1.0
Dead foetuses	N Mean SD	21 0 0.0	19 0 0.0	21 0 0.7	20 0 0.0
Post-implantation loss	N Mean SD	21 1 0.9	19 1 0.9	21 1 1.3	20 1 1.0
Post-implantation loss (%)	N Mean SD	21 8.1 9.27	19 7.9 8.95	21 8 12.64	20 9.6 23.15
Live foetuses	N Mean SD	21 10 2.1	19 10 1.6	21 10 2.5	20 10 3.4
Gravid uterine weight (g)	N Mean SD	21 68.9 12.60	19 70.5 10.70	21 67.9 16.20	19 69.0 16.13
Corrected body weight (carcass weight; g)	N Mean SD	21 243.8 22.73	19 252.1 16.00	21 247.3 17.42	19 246.0 19.47
Corrected weight change days 3 to 21 (g)	N Mean SD	21 49.0 8.8	19 51.1 9.43	21 51.3 8.72	19 50.7 11.38
Total weight change days to 21 (g)	N Mean SD	21 117.9 15.59	19 121.6 12.54	21 119.2 16.97	19 119.7 19.70
No. of females with live foetuses	N	21	19	21	20
Mean number of male foetuses per litter	N Mean SD	21 5 1.6	19 5 1.8	21 5 1.6	20 5 2.2
Mean number of female foetuses per litter	N Mean SD	21 5 2.1	19 5 1.5	21 5 1.9	20 5 2.0
% Male foetuses	N Mean SD	21 48 16.4	19 49 14.1	21 47 11.4	20 49 17.1
Mean foetal weight (g)	N Mean SD	21 5.31 0.465	19 5.37 0.264	21 5.29 0.381	19 5.18 0.399
Mean weight male foetuses (g)	N Mean Adj. mean SD	21 5.46 5.46 0.504	19 5.54 5.53 0.279	21 5.40 5.39 0.344	19 5.32 5.34 0.425
Mean weight female foetuses (g)	N Mean Adj. mean SD	21 5.13 5.13 0.424	19 5.22 5.22 0.274	21 5.19 5.18 0.465	19 5.01 5.03 0.412

*Number examined reduced due to excluded data

 

Table 3: Summary of Foetal Observations

		Dose Group (mg/kg)
		0	40	125	400
Malformation in foetal external - live
Number of dams/foetus		22/209	22/188	22/206	22/194
Number examined litter/foetus		21/209	19/188	21/206	19/194
Eye; eye bulge; small (malformation)	% Litter % Foetal	1/1 5 0.43	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00
Variation in foetal external - live
Tail; fleshy tab	% Litter % Foetal	1/1 5 0.43	2/3 11 1.61	1/1 5 0.43	0/0 0 0.00
Malformation in foetal fresh visceral - live
Number of dams/foetus		22/209	22/188	22/206	22/194
Number examined litter/foetus		21/105	19/96	21/103	19/97
Eye; anophthalmia	% Litter % Foetal	1/1 5 0.95	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00
General; situs inversus	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00	1/1 5 1.05
Thyroid; absent	% Litter % Foetal	1/1 5 0.95	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00
Gonad; small	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00	1/1 5 2.63
Variation in foetal fresh visceral - live
Number of dams/foetus		22/209	22/188	22/206	22/194
Number examined litter/foetus		21/105	19/96	21/103	19/97
Blood vessel; umbilical artery; left-sided	% Litter % Foetal	7/8 33 7.10	7/9 37 9.04	13/20 62 18.89	11/14 58 15.05
Kidney; dilated	% Litter % Foetal	0/0 0 0.00	1/1 5 0.88	0/0 0 0.00	0/0 0 0.00
Liver; liver lobe; discoloured	% Litter % Foetal	0/0 0 0.00	1/1 5 1.05	1/1 5 0.95	0/0 0 0.00
Liver; liver lobe; supernumerary	% Litter % Foetal	1/2 5 1.90	2/2 11 1.75	1/1 5 1.19	0/0 0 0.00
Thoracic cavity; fluid filled	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	1/1 5 0.95	0/0 0 0.00
Thymus; thymic remnant	% Litter % Foetal	0/0 0 0.00	1/1 5 1.05	2/2 10 1.75	0/0 0 0.00
Thyroid; small	% Litter % Foetal	1/1 5 0.95	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00
Ureter; dilated	% Litter % Foetal	0/0 0 0.00	2/2 11 2.19	1/1 5 1.19	0/0 0 0.00
Abdomen; abdominal cavity; fluid filled	% Litter % Foetal	4/6 19 5.00	1/1 5 0.88	3/3 14 2.82	0/0 0 0.00
Malformation in foetal fixed visceral - live
Number of dams/foetus		22/209	22/188	22/206	22/194
Number examined litter/foetus		21/104	19/92	21/103	19/97
Diaphragm; hole	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00	1/1 5 0.75
Eye; macrophthalmia	% Litter % Foetal	2/3 10 3.97	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00
Eye; microphthalmia	% Litter % Foetal	1/1 5 1.19	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00
Variation in foetal fixed visceral - live
Number of dams/foetus		22/209	22/188	22/206	22/194
Number examined litter/foetus		21/104	19/92	21/103	19/97
Blood vessel; umbilical artery; left-sided	% Litter % Foetal	9/14 43 13.55	7/11 37 11.40	10/15 48 13.46	10/21 53 21.23
Brain; brain ventricle; dilated	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00	1/1 5 1.05
Eye; lens; altered texture	% Litter % Foetal	1/1 5 1.59	0/0 0 0.00	0/0 0 0.00	2/2 11 2.37
Eye; retina; fold	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	1/1 5 0.68	0/0 0 0.00
Kidney; dilated	% Litter % Foetal	3/3 14 4.29	5/9 26 9.47	11/17 52* 18.90	6/7 32 6.29
Liver; liver lobe; supernumerary	% Litter % Foetal	5/5 24 4.41	8/9 42 10.18	4/4 19 4.81	7/9 37 9.09
Thymus; thymic remnant	% Litter % Foetal	2/2 10 1.90	4/6 21 5.79	3/3 14 2.43	7/7 37* 7.29
Ureter; dilated	% Litter % Foetal	1/1 5 2.38	1/1 5 1.05	0/0 0 0.00	1/1 5 0.75
Malformation in foetal skeletal - live
Number of dams/foetus		22/209	22/188	22/206	22/194
Number examined litter/foetus		21/105	19/96	21/103	19/97
Skull; mandible; fused	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	1/1 5 0.95	0/0 0 0.00
Skull; mandible; single incisor socket	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	1/1 5 0.95	0/0 0 0.00
Skull; orbital socket; small	% Litter % Foetal	1/1 5 0.95	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00
Skull; palatine; cleft	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	1/1 5 0.95	0/0 0 0.00
Vertebra; cervical arch; absent	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00	1/1 5 1.05
Vertebra; cervical arch; small	% Litter % Foetal	1/1 5 0.95	0/0 0 0.00	0/0 0 0.00	1/1 5 1.05
Vertebra; cervical centrum; absent	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00	1/1 5 1.05
Vertebra; thoracic centrum; hemicentric	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	1/1 5 0.79	1/1 5 1.05
Rib; abnormal attachment	% Litter % Foetal	1/1 5 0.95	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00
Rib; branched	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00	1/1 5 1.05
Rib; fused	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00	1/1 5 1.05
Variation in foetal skeletal - live
Number of dams/foetus		22/209	22/188	22/206	22/194
Number examined litter/foetus		21/105	19/96	21/103	19/97
Skull; frontal; incomplete ossification	% Litter % Foetal	1/1 5 1.19	0/0 0 0.00	1/1 5 0.79	0/0 0 0.00
Skull; frontal; unossified line	% Litter % Foetal	1/1 5 0.79	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00
Skull; hyoid; incomplete ossification	% Litter % Foetal	1/1 5 0.95	1/1 5 0.88	1/2 5 1.59	0/0 0 0.00
Skull; hyoid; unossified	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	1/1 5 0.95	0/0 0 0.00
Skull; interparietal; incomplete ossification	% Litter % Foetal	5/8 24 8.33	2/2 11 1.93	4/6 19 5.08	6/10 32 9.24
Skull; parietal; incomplete ossification	% Litter % Foetal	3/7 14 6.98	2/3 11 3.63	3/7 14 5.87	6/10 32 9.27
Skull; parietal; unossified line	% Litter % Foetal	1/1 5 0.79	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00
Skull; presphenoid; incomplete ossification	% Litter % Foetal	1/1 5 0.95	0/0 0 0.00	1/1 5 0.95	0/0 0 0.00
Skull; squamosal; incomplete ossification	% Litter % Foetal	2/3 10 2.86	0/0 0 0.00	2/2 10 1.75	1/1 5 0.88
Skull; supraoccipital; incomplete ossification	% Litter % Foetal	3/3 14 3.10	0/0 0 0.00	2/3 10 2.54	3/3 16 3.12
Skull; suture; sutural bone	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	2/2 10 1.90	½ 5 2.11
Skull; zygomatic arch; incomplete ossification	% Litter % Foetal	2/3 10 3.10	2/2 11 1.93	2/3 10 2.54	0/0 0 0.00
Skull; zygomatic arch; ossification bridge	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	1/1 5 0.79	0/0 0 0.00
Pelvic girdle; iliac alignment; abnormal	% Litter % Foetal	9/19 43 20.16	10/21 53 21.67	7/13 33 11.92	5/9 26 8.33
Sternebra; dumbbell ossification	% Litter % Foetal	0/0 0 0.00	1/1 5 1.05	0/0 0 0.00	1/1 5 1.05
Sternebra; incomplete ossification	% Litter % Foetal	10/18 48 15.87	5/6 26 6.05	8/16 38 14.57	7/13 37 12.82
Sternebra; increased ossification	% Litter % Foetal	0/0 0 0.00	1/1 5 0.88	2/2 10 1.75	0/0 0 0.00
Sternebra; misaligned ossification centres	% Litter % Foetal	1/1 5 1.59	1/1 5 1.05	2/2 10 1.75	1/1 5 1.05
Sternebra; unossified	% Litter % Foetal	1/1 5 0.95	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00
Supernumerary rib; present	% Litter % Foetal	21/85 100 80.04	19/65 100 69.47	20/63 95 61.94	17/54 89 53.78
Vertebra; cervical arch; incomplete ossification	% Litter % Foetal	2/2 10 2.14	0/0 0 0.00	1/1 5 1.19	3/3 16 2.81
Vertebra; cervical centrum; incomplete ossification	% Litter % Foetal	9/19 43 17.94	8/10 42 11.32	10/18 48 17.14	13/23 68 21.67
Vertebra; cervical centrum; misaligned	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00	1/1 5 1.05
Vertebra; cervical centrum; unilateral ossification	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00	1/1 5 1.05
Vertebra; cervical centrum; unossified	% Litter % Foetal	12/22 57 21.75	7/10 37 11.58	11/24 52 22.98	7/14 37 13.07
Vertebra; lumbar centrum; asymmetric ossification	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	1/1 5 1.19	1/1 5 0.75
Vertebra; lumbar centrum; bipartite ossification	% Litter % Foetal	1/1 5 1.19	0/0 0 0.00	2/2 10 1.98	0/0 0 0.00
Vertebra; lumbar centrum; dumbbell ossification	% Litter % Foetal	1/3 5 3.57	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00
Vertebra; lumbar centrum; incomplete ossification	% Litter % Foetal	1/1 5 0.95	1/1 5 1.05	1/1 5 1.19	1/1 5 0.75
Vertebra; sacral arch; ossification bridge	% Litter % Foetal	5.7 24 6.98	4/8 21 7.89	10/16 48 15.48	7/10 37 11.84
Vertebra; sacral centrum; incomplete ossification	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00	1/1 5 1.05
Vertebra; thoracic centrum; bipartite ossification	% Litter % Foetal	1/2 5 2.38	0/0 0 0.00	1/1 5 1.19	1/1 5 1.05
Vertebra; thoracic centrum; dumbbell ossification	% Litter % Foetal	2/3 10 3.17	3/3 16 2.81	2/2 10 1.75	1/1 5 1.05
Vertebra; thoracic centrum; incomplete ossification	% Litter % Foetal	2/3 10 3.97	1/1 5 1.05	4/6 19 5.40	2/2 11 1.63
Vertebra; thoracic centrum; misaligned	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	1/1 5 0.79	1/1 5 1.05
Forelimb; phalanx; incomplete ossification	% Litter % Foetal	2/3 10 2.86	0/0 0 0.00	2/4 10 3.33	3/4 16 4.26
Forelimb; phalanx; unossified	% Litter % Foetal	1/2 5 1.90	1/1 5 1.05	2/2 10 2.14	0/0 0 0.00
Hindlimb; metatarsal; incomplete ossification	% Litter % Foetal	5/7 24 6.39	6/11 32 10.00	4/4 19 3.54	7/13 37 11.87
Hindlimb; metatarsal; unossified	% Litter % Foetal	5/9 24 8.89	2/2 11 2.19	5/8 24 7.86	4/6 21 6.63
Hindlimb; phalanx; incomplete ossification	% Litter % Foetal	14/30 67 27.14	14/36 74 37.98	15/30 71 28.73	14/34 74 33.55
Hindlimb; phalanx; unossified	% Litter % Foetal	10/29 48 26.27	12/27 63 26.32	10/28 48 24.37	11/33 58 31.23
Rib; clubbed	% Litter % Foetal	0/0 0 0.00	0/0 0 0.00	0/0 0 0.00	1/1 5 1.32
Rib; nodulated	% Litter % Foetal	2/3 10 2.70	2/3 11 2.63	3/3 14 2.78	1/1 5 1.05

*Fisher 1-tail Ascending Test significant at the 0.05 level

Table 4: Selected Haematology Parameters

Dose Group (mg/kg)	0	40	125	400
Haemaglobin (g/dL)	11.2 ± 0.50	11.1 ± 0.47	11.0 ± 0.63	10.6 ± 0.59*
Red blood cell count (10^12/L)	6.00 ± 0.204	5.91 ± 0.274	5.92 ± 0.311	5.72 ± 0.310
Packed cell volume (%)	34.4 ± 1.46	33.6 ± 1.34	33.5 ±1.85	32.9 ± 1.89
Reticulocytes (%)	3.1 ± 0.72	3.3 ± 0.87	2.7 ± 0.93	4.5 ± 1.53*

* p<0.05

Table 5: Selected Clinical Biochemistry Parameters

Dose Group (mg/kg)	0	40	125	400
Aspartate aminotransferase (IU/L)	55 ± 6.3	61 ± 11.5	67 ± 19.0	88 ± 42.0*
Alanine aminotransferase (IU/L)	52 ± 9.4	58 ± 9.3	58 ± 5.0	69 ± 7.5**

* p<0.01

** p<0.001

Table 6: Summary of Organ weights

Dose Group (mg/kg)	0	40	125	400
Terminal body weight (g)	313.1 ± 31.98	322.8 ± 18.39	315.1 ± 29.87	314.0 ± 27.22
Spleen (absolute) (g)	0.493 ± 0.0918	0.560 ± 0.0943	0.522 ± 0.0635	0.599 ± 0.0486*
Spleen (% body weight)	0.157 ± 0.0217	0.174 ± 0.0276	0.166 ± 0.0163	0.192 ± 0.0269*

* p<0.01

Conclusions:

Based on the findings in this study, the female No Observed Adverse Effect Level (NOAEL) is considered to be 125 mg/kg/day, and foetal NOAEL is considered to be >=400 mg/kg/day.

Executive summary:

A study was conducted to determine the effects of the test material on the embryonic and foetal development of the rat in accordance with the standardised guidelines OECD 414, US EPA OPPTS 870.3700, the study was carried out under GLP conditions.

Four groups of 22 female time-mated Crl:WI(Han) rats were administered 0 (control article [vehicle]), 40, 125, or 400 mg/kg/day 4-OH TEMPO orally by gavage daily from Gestation Day (GD) 6 to 20, inclusive, at a volume of 10 mL/kg. The control article (vehicle) was Polyethylene glycol 400 (PEG 400).

Assessment of toxicity was based on evaluation of clinical observations, body weights, food consumption, and clinical pathology. On GD 21, females were sacrificed, and the progress and outcome of pregnancy were evaluated. Complete necropsies were performed on all animals, with a recording of macroscopic abnormalities for all tissues. Organ weights were conducted as indicated. Fetuses were evaluated for external, visceral, and skeletal malformations and variations.

Statistically significantly, but marginally decreased haemoglobin and increased reticulocytes were noted in animals administered the top dose of 400 mg/kg/day. These were correlated with a statistically significant increase in spleen weights. These changes, while attaining statistical significance are not considered to be toxicologically significant.

These were correlated with a statistically and toxicologically significant increase in spleen weights, and were indicative of toxicologically significant anaemic processes, showing the top dose chosen had been adequate. There were also marginal increases in aspartate- and alanine aminotranseferase activities, although in the absence of any liver weight findings, they are not considered intrinsically adverse in nature. 

No adverse effects on bodyweight, food consumption, clinical or postdose observations, macroscopic findings, or reproductive parameters were noted.

Based on the findings in this study the female No Observed Adverse Effect Level (NOAEL) is considered to be 125 mg/kg/day, and foetal NOAEL is considered to be 400 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A study was conducted to determine the effects of the test material on the embryonic and foetal development of the rat in accordance with the standardised guidelines OECD 414, US EPA OPPTS 870.3700, the study was carried out under GLP conditions.

Four groups of 22 female time-mated Crl:WI(Han) rats were administered 0 (control article [vehicle]), 40, 125, or 400 mg/kg/day 4-OH TEMPO orally by gavage daily from Gestation Day (GD) 6 to 20, inclusive, at a volume of 10 mL/kg. The control article (vehicle) was Polyethylene glycol 400 (PEG 400).

Assessment of toxicity was based on evaluation of clinical observations, body weights, food consumption, and clinical pathology. On GD 21, females were sacrificed, and the progress and outcome of pregnancy were evaluated. Complete necropsies were performed on all animals, with a recording of macroscopic abnormalities for all tissues. Organ weights were conducted as indicated. Fetuses were evaluated for external, visceral, and skeletal malformations and variations.

Statistically significantly, but marginally decreased haemoglobin and increased reticulocytes were noted in animals administered the top dose of 400 mg/kg/day. These were correlated with a statistically significant increase in spleen weights. These changes, while attaining statistical significance are not considered to be toxicologically significant.

These were correlated with a statistically and toxicologically significant increase in spleen weights, and were indicative of toxicologically significant anaemic processes, showing the top dose chosen had been adequate. There were also marginal increases in aspartate- and alanine aminotranseferase activities, although in the absence of any liver weight findings, they are not considered intrinsically adverse in nature. 

No adverse effects on bodyweight, food consumption, clinical or postdose observations, macroscopic findings, or reproductive parameters were noted.

Based on the findings in this study the female No Observed Adverse Effect Level (NOAEL) is considered to be 125 mg/kg/day, and foetal NOAEL is considered to be 400 mg/kg/day.

Justification for classification or non-classification

No classification for reproduction is proposed based on the available data. There were no adverse effects on reproduction as examined in the OECD 414 study with rats.