1,4-Benzenediamine, 2-(methoxymethyl)-

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

other: the study was performed with the sulfate salt of 1,4-diamino-2-methoxymethylbenzene

Adequacy of study:

key study

Study period:

From May 04, 2006 to Sept. 25, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Remarks:

according to OECD Principles of GLP and Principles of GLP described in the French Official Journal of 18 September 2004

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

337906-37-3

Cas Number:

337906-37-3

IUPAC Name:

337906-37-3

Constituent 2

Reference substance name:

-

EC Number:

474-270-7

EC Name:

-

IUPAC Name:

474-270-7

Constituent 3

Reference substance name:

1,4-Diamino-2-methoxymethyl-benzene sulfate (1:1)

IUPAC Name:

1,4-Diamino-2-methoxymethyl-benzene sulfate (1:1)

Details on test material:

- Name of test material: 1,4-diamino-2-methoxymethyl-benzene sulfate (1: 1), WR 801337, (Methoxymethyl-PPD-Sulfat.) (code # A012220)
- TSIN: 801337
- Molecular formula (if other than submission substance): C8C12N2OxH2SO4
- Molecular weight (if other than submission substance): 250.28
- Substance type: Pure active substance
- Physical state: Cream-rosy powder- Stability: The substance on storage in dryness and darkness is considered to be stable for more than 8 years.
- Stability in Solution: Stable for seven days in water, DMSO, water and water/acetone 1:1 at room temperature
- Storage condition of test material: At room temperature, protected from light and moisture
- Solubility: > 10 weight% in water (pH 1.75); 0.03 weight% in acetone; 1.7 weight% in acetone/water (1:1); 15 weight% in DMSO

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sprague-Dawley rats Crl: OFA.SD were obtained from Charles River Laboratories, Domaine des Oncins, 69210 Saint Germain sur l'Arbresle, France
- Age at study initiation: 10-13 wk
- Weight at study initiation: 205-263 g
- Fasting period before study: No
- Housing: Individually, in plastic cages (dimensions 365 x 225 x 180 mm) with autoclaved sawdust as bedding; Air conditioned room in a barrier protected unit
- Diet: Pelleted commercial complete rodent diet (Diet reference A04C-10), ad libitum (Diets were sterilised by irradiation and analysed for chemical and bacterial contaminants)
- Water: Filtered (0.2 µm) mains drinking water, ad libitum (via bottles)
- Acclimation period: 6 d
- Periodical analysis of diet, drinking water or bedding revealed no contamination at levels which might have interfered with achieving the objective of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22± 2°C
- Humidity: At least 40 % (Relative)
- Air changes: At least 15 air changes per h
- Photoperiod: 12 h light/12 h dark cycle

IN-LIFE DATES: From: May 15, 2006 To: May 29, 2006

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.4 % ascorbic acid in water for injection

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was prepared as a solution in the vehicle at concentrations of 2, 6 and 18 mg/mL. The final formulations were adjusted to pH 5.1 ± 0.2 (range 4.92 to 5.27) by addition of NaOH 1N (prepared at the Testing Facility by dilution of Titrisol supplied by Laboratoire Merck, Munchen, Germany in water for injection supplied by Laboratoire Aguettant, Lyon, France).
- Frequency of preparation: Daily

VEHICLE:
- Justification for use and choice of vehicle: Not reported
- Concentration in vehicle: 0, 2, 6 and 18 mg/mL
- Amount of vehicle: 5 mL (individual dose volumes were adjusted on the days that the animals were weighed)
- Lot/batch no.: Water: Laboratoire Aguettant, Lyon, France; Batch # Not reported; Ascorbic acid: Sigma-Aldrich, Steinheim, Germany
- Storage: Ascorbic acid: Refrigerated (approx. 4°C); Water: At room temperature
- Stability: 9 d

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate samples 5 mL each from each formulation and controls were taken on the first and last days of dosing. Samples were stored at approx. -20°C, protected from light and sent packed in dry ice to sponsor for analysis. According to the sponsor analytical report, the test material Methoxymethyl-PPD-Sulfate (18 %, w/v) is distributed homogeneously in aqueous ascorbic acid solution. Consequently, it was assumed that formulations of lower dye levels are in homogeneous mode as well. Analysis of the low, intermediate and high dose samples and backup samples (85.1 - 91.5 %) on Day G6 and G19 are not distributed statistically across the acceptable range of deviation (90-110 %) but they are very close above its lower limit.

Details on mating procedure:

The female rats were mated at the supplier facility with a documented day of mating. They were received at the testing facility on Day 0 of gestation.

Duration of treatment / exposure:

From Day 6 to 19 of gestation

Frequency of treatment:

Once daily

Duration of test:

20 d

No. of animals per sex per dose:

25 females

Control animals:

other: yes, water for injection

Details on study design:

- Dose selection rationale: The dose levels of 10, 30 and 90 mg/kg bw/day given by the oral route with a volume of administration of 5 mL/kg were chosen based on the results of a preliminary study in pregnant rats (MDS study# AA28648)
- Rationale for animal assignment: Performed at arrival of animals to the test facility using computer-generated random numbers.
Justification for use of testing animal: Rodent species acceptable to the regulatory agencies. Background data for the strain were available at the testing facility.
Rationale for choice of route of administration: The oral route was selected as this was considered a possible route of human exposure during manufacture, handling or use of the test material.

Examinations

Maternal examinations:

MORBIDITY/MORTALITY: Yes
- Time schedule: At least twice daily

CLINICAL OBSERVATIONS: Yes (Abnormalities in appearance, behavior and clinical signs of reaction to treatment.)
- Time schedule: Once before and at least once after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, 6, 9, 12, 15, 18 and 20 of gestation

FOOD CONSUMPTION: Yes
- Individual food consumption was measured for the periods (days): 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20 of gestation.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day # 20 (were euthanised by carbon dioxide inhalation)
- Organs examined: Females were dissected and examined for macroscopic pathological changes and abnormal organs were sampled and preserved in 10% neutral formalin. The ovaries and uterus of each female were removed and examined

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 
- Individual fetal weights and sex: Yes
- Other: Uterus of all females was placed in ammonium sulphide solution in order to stain any previously undetected implantation sites.

Fetal examinations:

- External examinations: Yes: [all per litter] 
- Soft tissue examinations: Yes: [all per litter] (half of each litter was examined for visceral anomalies, remaining fetuses were preserved in Harrisson's fluid for fixed visceral examination) 
- Skeletal examinations: Yes: [half per litter] (following maceration of the soft tissues in potassium hydroxide solution, ossified skeleton was stained with Alizarin red and preserved in glycerol) 
- Head examinations: No data
- Method of skeletal and fixed-visceral examinations: Performed under low power magnification
- Following external examination fetus were euthanised by an intraperitoneal injection of sodium pentobarbitone.

Statistics:

Statistical analysis was performed by the data acquisition software (TASC) where appropriate, as follows: The data were checked for homogeneity of variance across groups using Bartlett's test. Homogenous data were then analysed by parametric methods, i.e. ANOVA followed by Dunnett's test if ANOVA was significant. Non-homogenous data were analysed by non-parametric methods, i.e. Kruskal-Wallis test followed by Dunn's test if the Kruskal-Wallis was significant. The numbers of resorptions, number of dead fetuses and all litter-based percentages were analysed using the above non-parametric methods.

Indices:

- Following parameters were calculated for each group:  
- Pre-implantation loss (%) = ((number of corpora lutea - number of implantations)/number of corpora lutea)x100
- Post-implantation loss (%) = ((number of implantations - number of viable fetuses)/number of implantations)x100

Historical control data:

The background data on the following parameters were presented in the report: maternal body weight gain (g) during gestation, maternal food consumption (g/day) during gestation, caesarean data collected on day 20 of gestation, caesarean data collected on Day 20 of gestation, incidence of malformations (external, internal & skeletal), fetal examination – external, fetal examination - fixed soft tissue and fetal examination – skeletal.
 
Details are provided in the study report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
- There was no unscheduled death in any group. At 30 mg/kg bw/day and at 90 mg/kg bw/day, orange discoloration of the urine was noted
-Mean body weight gain and mean food consumption was statistically significantly reduced in the high dose group during the first 3 days of treatment (Days 6 to 9 of gestation) compared with the controls. Thereafter, mean body weight gain was comparable with, or slightly greater than, that in the controls and from Day 12 of gestation through to termination, food consumption was comparable too through to termination. Mean terminal body weight was comparable with that of the control animals.
- No treatment-related macroscopic changes were noted at necropsy of the adult females in any group. There was no adverse effect of treatment on mean uterus weight in any group.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: Transient reduction in mean body weight gain and food consumption

2

Dose descriptor:

NOAEL

Effect level:

90 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: overall effects

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: No treatment related toxicity was observed

Details on embryotoxic / teratogenic effects:
-There were 22, 24, 25 and 25 pregnant females in the control, low, mid and high dose groups, respectively at the terminal caesarean sections. The pre-implantation data (mean numbers of corpora lutea and implantation sites and the corresponding percentage pre-implantation loss) were comparable in all groups
- There was no obvious adverse effect of treatment on embryo-foetal survival in any group. There was a slightly higher incidence of late resorptions in the high dose group compared with the control group. This was related due to a single female with 5 late resorptions. This isolated finding was considered to be incidental. Another female in the high dose group showed one late resorption and no viable foetuses. This incidental finding was considered to be a normal physiological response due to the presence of the single implantation and of no toxiclogical relevance.
-Mean live litter size was consequently comparable in all groups. Mean foetal weight and sex ratio were comparable in all groups.
-There was no external abnormality noted in any group. The only visceral malformation noted was marked renal pelvic dilatation for one control fetus. The incidences of other less severe soft tissue anomalies and variations, which principally included slight renal pelvic dilatation and convoluted and slightly dilated ureters, did not suggest any influence of treatment and are commonly observed in rats.
-There was no skeletal malformation noted in any group. The incidences of foetuses with delayed ossification of the cranium (parietal, interparietal and squamosal bones), paws (metacarpal of the 2nd or 5th digits) or sternum (2nd/4th and 6th sternebra) were slightly higher in the 90 mg/kg bw/day group by comparison with the incidence both the in controls and the historical control data. A similar but less marked increase was observed in the 30 and 10 mg/kg bw/day groups (affecting the cranium and paws only). None of these findings was statistically significant. These findings were indicative of a minor delay in foetal ossification. Minor delays in ossification are often seen as secondary maternal related effects, are not mechanistically linked to malformation, and are not generally considered adverse (s. Carney, E. W.; Kimmel.: C. A.: Interpretation of Skeletal Variations for Human RiskAssesment: Delayed Ossification and Wavy Ribs; Birth Def. Res. (Part B): Vol. 80, 473-496, 2007). Therefore, this finding was considered of no toxicological relevance. There was also a slightly greater incidence of rudimentary 14th ribs in all the treated groups compared with the incidence in the control animals. Such findings in rodent studies are considered to be associated with maternal stress (s. Chernoff, N.; Rogers, J. M.: Supernumerary ribs in developmental toxicity bioassays and in human populations: Incidence and biological significance; J. Toxicol. Enivon. Health,Part B: Vol. 7, 437-449, 2004). Furthermore, these findings are of no known physiological consequence and were therefore, considered to be of no toxicological relevance.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 90 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: overall effects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Key result

Developmental effects observed:

no

Treatment related:

no

Dose response relationship:

no

Relevant for humans:

no

Applicant's summary and conclusion

Conclusions:

Administration of Methoxymethyl-PPD-Sulfat by the oral route during organogenesis (Day 6 to 19 of gestation) at dose level of 0, 10, 30 and 90 mg/kg bw/day resulted in NOAEL of 30 mg/kg bw/day for maternal toxicity (based on a transient reduction in mean body weight gain and food consumption ). The NOAEL: 30 mg/kg/bw for methoxymethyl-PPD-Sulfate, corresponds to 30 x 0.61 = 18.3 mg/kg bw for methoxymethyl-PPD-base using the conversion factor of 0.61. The NOAEL for developmental toxicity was 90 mg/kg bw/day for sulfate and corresponds to 90 x 0.61 = 55 mg/kg bw for methoxymethyl-PPD-base.

Executive summary:

The developmental toxicity study of 1,4-diamino-2-methoxymethyl-benzene sulfate (1: 1), (Methoxymethyl-PPD-Sulfat.) was determined following OECD guideline 414 (Prenatal Developmental Toxicity Study).

The purpose of this study was to assess the effects of 2-METHOXY-METHYL-PPHENYLENEDIAMINESULFATE on pregnant female rats and embryo-foetal developmentwhen administered orally, by gavage, once daily to mated female rats from Day 6 through to Day19 post coitum, inclusive. Each group consisted of 25 mated female rats.2-METHOXYMETHYL- P-PHENYLENEDIAMINE SULFATE was administered once daily by gavage at doselevels of 10, 30 and 90 mg/kg bw/day. Vehicle control animals were dosed with the vehiclealone (0.4% ascorbic acid dissolved in water for injection). Animals received a standard dose volumeof 10 mL/kg bw with adjustment to the actual body weight on days the animals were weighed.Test solutions were prepared freshly daily. Samples from the formulations were analyzed foractual concentrations and homogeneity and stability of the test item at the first and the last day ofdosing. The samples from Day G6 were found to be within a range of 85.1 - 91.5 % of the nominal concentration (low dose 85.1%, mid dose 89.1% and high dose 91.5%). The samples of Day G19 were found to be within a range of 90.2 – 91.0%. As the deviation in the mid dose (maternal NOAEL) from the measurement on Day G6 is near of the required 90.0% lower range of acceptance, and the second measurement at Day G19 was found to be within the range of ±10%, no dose corrections was made.

Food consumption data were calculated for the periods from Day 0 to 3, 3 to 6, 6 to 9, 9 to12, 12 to 15, 15 to 18, and 18 to 20 of gestation. Body weights were recorded on Days 0, 6, 9, 12, 15, 18, and 20 of gestation. All animals were checked at least twice daily for clinical signs. At Day 20 of gestation, all mated females were sacrificed by CO2-asphyxiation and a complete autopsy and a macroscopic examination of the organs was carried out. Post mortem examination, including gross macroscopic examination of all internal organs, with emphasis on the uterus, uterine contents, position of foetuses in the uterus and number of corpora lutea, was performed and the data were recorded. The intact uterus (prepared by Caesarean section) was removed and the percentage of resorption sites (early, late) and foetuses (live or dead) as well as their uterine position were recorded. In addition, uterine weights were determined. The number of implantation sites (both before and after staining with aqueous ammonium sulfide) and corpora lutea were also determined.

Each viable foetus was weighed, sexed and examined for gross external malformations and euthanized. Half of the foetuses were prepared for the micro-dissection technique with fixation in Harrison´s fluid to examine the soft tissues. The remaining foetuses were eviscerated and processed for the cartilage (Alcian blue) and skeletal (Alizarin red S) staining to examine the skeleton and cartilage.

There was no unscheduled death in any group. At 30 mg/kg bw/day and at 90 mg/kg bw/day, orange discoloration of the urine was noted Mean body weight gain and mean food consumption was statistically significantly reduced in the high dose group during the first 3 days of treatment (Days 6 to 9 of gestation) compared with the controls. Thereafter, mean body weight gain was comparable with, or slightly greater than, that in the controls and from Day 12 of gestation through to termination, food consumption was comparable too through to termination. Mean terminal body weight was comparable with that of the control animals. No treatment-related macroscopic changes were noted at necropsy of the adult females in any group. There was no adverse effect of treatment on mean uterus weight in any group.

There were 22, 24, 25 and 25 pregnant females in the control, low, mid and high dose groups, respectively at the terminal caesarean sections. The pre-implantation data (mean numbers of corpora lutea and implantation sites and the corresponding percentage pre-implantation loss) were comparable in all groups

There was no obvious adverse effect of treatment on embryo-foetal survival in any group. There was a slightly higher incidence of late resorptions in the high dose group compared with the control group. This was related due to a single female with 5 late resorptions. This isolated finding was considered to be incidental. Another female in the high dose group showed one late resorption and no viable foetuses. This incidental finding was considered to be a normal physiological response due to the presence of the single implantation and of no toxiclogical relevance.

Mean live litter size was consequently comparable in all groups. Mean foetal weight and sex ratio were comparable in all groups. There was no external abnormality noted in any group. The only visceral malformation noted was marked renal pelvic dilatation for one control fetus. The incidences of other less severe soft tissue anomalies and variations, which principally included slight renal pelvic dilatation and convoluted and slightly dilated ureters, did not suggest any influence of treatment and are commonly observed in rats.

There was no skeletal malformation noted in any group. The incidences of foetuses with delayed ossification of the cranium (parietal, interparietal and squamosal bones), paws (metacarpal of the 2^nd or 5^th digits) or sternum (2^nd/4^th and 6^th sternebra) were slightly higher in the 90 mg/kg bw/day group by comparison with the incidence both the in controls and the historical control data. A similar but less marked increase was observed in the 30 and 10 mg/kg bw/day groups (affecting the cranium and paws only). None of these findings was statistically significant. These findings were indicative of a minor delay in foetal ossification. Minor delays in ossification are often seen as secondary maternal related effects, are not mechanistically linked to malformation, and are not generally considered adverse. Therefore, this finding was considered of no toxicological relevance. There was also a slightly greater incidence of rudimentary 14th ribs in all the treated groups compared with the incidence in the control animals. Such findings in rodent studies are considered to be associated with maternal stress. Furthermore, these findings are of no known physiological consequence and were therefore, considered to be of no toxicological relevance.

Based on a transient reduction in mean body weight gain and food consumption in the 90 mg/kg bw/day group, the no observed adverse effect level (NOAEL) for maternal toxicity was considered to be 30 mg/kg bw/day. The NOAEL for developmental toxicity was concluded to be 90 mg/kg bw/day.

This developmental toxicity study is classified as acceptable, and satisfies the guideline requirements of the OECD 414 method.