Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
calculation (if not (Q)SAR)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Based on results of reliable (K1) studies from section 7.6.2 genetic toxicity in vivo on both rats and mice
Justification for type of information:
Expert judgment based on result from supporting substance (structural analogue or surrogate) MBB sulphate
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004
Reference Type:
other company data
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Principles of method if other than guideline:
To determine the acute oral LD50 for the calculations herein, the MTD of the sulfate salt from two genotoxicity studies were applied. The conditions of these in vivo studies were comparable to those recommended
in the OECD 423 guideline. Based on these studies, the LD50 value of the sulfate salt was determined to be 100-200 mg/kg bw. For the calculations of the acute dermal and inhalation toxicity, an oral LD50 of 150 mg/kg bw for the sulfate salt was used for the route extrapolation calculations. The rationale for this is as follows:
‐ The same number of animals was treated as the minimal number required in the OECD guideline for the acute toxicity studies.
‐ The derived LD50 value of 100-200 mg/kg bw for the sulfate salt is within the required test concentrations and possible LD50 estimations, namely between 50 and 300 for the OECD.
‐ In the case of 2-METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE, the observation period of two days was considered as relevant, because the major elimination of the parent compound and its metabolites was within 48 hours, as demonstrated in the ADME study (Ref 5): “After intravenous and low oral dosing the highest rate of excretion was observed during the first 8 hours (between 60 and 64% of total urine excretion). Thereafter, a decreasing excretion rate was noted with increasing time intervals. In the high oral and high dermal group the excretion was more evenly divided over the 0-8 and 8-24 h interval. At termination of the study, the average total remaining radioactivity in blood,
carcass plus tissues was between 0.9% and 1.7% of the administered dose in the intravenous, oral and dermal groups, indicating no major accumulation of radioactivity after 72 hours.”
‐ In addition, no cumulative toxic effect of 2-METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE is expected. This is based on the fact that no animal died due to toxic effects in either the 90- day repeated dose study or the prenatal developmental study (in which the test item was administered in both studies daily) at the highest dose of 90 mg/kg bw.
‐ The LD50 for the sulfate salt (150 mg/kg bw) was used, since the in vivo toxicity and kinetics assays used in the calculations were also carried out with the sulfate salt.
The conversion factor of 0.61 was used to convert the oral LD50 of the sulfate salt to the free base. A GLP-compliant amendment for the Comet Assay and the UDS is in preparation, in which it will be stated, that the MTD determination was in line with the requirements of the OECD guideline 423 for acute oral toxicity testing.
Test type:
other: data available from the maximun tolerated dose after application from in vivo genotoxicity studies.

Test material

Constituent 1
Reference substance name:
1.4-diamino-2-methoxymethyl-benzene sulphate 1:1
IUPAC Name:
1.4-diamino-2-methoxymethyl-benzene sulphate 1:1
Constituent 2
Reference substance name:
Methoxymethyl-PPD-Sulfat
IUPAC Name:
Methoxymethyl-PPD-Sulfat
Details on test material:
- Name of test material: 1,4-diamino-2-methoxymethyl-benzene sulfate (1: 1), (Methoxymethyl-PPD-Sulfate), WR801337 (substance code: A012220)
- TSIN: 801337
- Substance type: Pure active substance
- Physical state: Cream-rosy powder
- Storage condition of test material: At room temperature

Test animals

Details on test animals or test system and environmental conditions:
Genetic toxicity (invivo)_71339_C337906-37-3_E474-270-7_QCE08June11
test animal: mouse (NMRI, both male and female)

Genetic toxicity in vivo (UDS)_60029_C337906-37-3_E474-270-7_QCE08June11
test animal: rat (Wistar Hanlbm: WIST (SPF), male)

Genetic toxicity in vivo (comet assay)_C337906-37-3_E474-270-7_QCE15June11
test animal: rat (CRL:(WI) BR Wistar, male)

For more details on test animals and environmental condition see the corresponding studies in the genetic toxicity in vivo section

Administration / exposure

Details on oral exposure:
Genetic toxicity (invivo)_71339_C337906-37-3_E474-270-7_QCE08June11
Dose Range Finding: In a pre-experiment two female mice received a single dose of 2000 mg/kg bw of 2-METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE intraperitoneally, dissolved in distilled water as vehicle. The volume administered was 10 ml/kg bw.
Main Experiment: A single dose of 100 mg/kg bw was administered by intraperitoneal injection in the main experiment. The volume administered was 10 ml/kg bw.

Genetic toxicity in vivo (UDS)_60029_C337906-37-3_E474-270-7_QCE08June11
test animal: rat (Wistar Hanlbm: WIST (SPF), male)
Dose Range finding: In the dose-range finding experiments 2-METHOXY-METHYL-PPHENYLENEDIAMINE SULFATE was administered in a single oral dose to two male and two female rats per group at doses of 100 (first preexperiment), 500 (second pre-experiment), 200 (third pre-experiment) and finally 150 mg/kg bw (fourth pre-experiment). The volume administered was 10 ml/kg bw. The vehicle was deionised water.
Main Experiment: In the main experiment, a single dose of 75 or 150 mg/kg bw 2- METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE was administered by oral gavage to four male rats per dose and observation period followed by a 4 or 16 hour observation period. The volume administered was 10 ml/kg bw. The vehicle was deionised water.

Genetic toxicity in vivo (comet assay)_C337906-37-3_E474-270-7_QCE15June11
test animal: rat (CRL:(WI) BR Wistar, male)
Dose Range Finding: A dose of 2000 mg/kg bw of 2-METHOXY-METHYL-PPHENYLENEDIAMINE SULFATE was administered to three male rats by oral gavage. The dose volume was 20 ml/kg bw. The vehicle was deionised water with 0.4% ascorbic acid. A second dose of 100 mg/kg bw 2-METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE was administered 20 hrs following the first dose.
Main Experiment: In the main experiment, 2-METHOXY-METHYL-PPHENYLENEDIAMINE SULFATE was administered by oral gavage twice at 20 hour intervals at doses of 25, 50 and 100 mg/kg bw to five male rats in each dose group. The dose volume was 20 ml/kg bw.

For more details see the corresponding studies in the genetic toxicity in vivo section

Results and discussion

Effect levelsopen allclose all
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 100 - < 200 mg/kg bw
Based on:
test mat.
Remarks:
sulfate salt
Remarks on result:
other: oral gavage (rats)
Key result
Sex:
not specified
Dose descriptor:
approximate LD50
Effect level:
ca. 91.5 mg/kg bw
Based on:
other: calculation using conversion facto
Remarks:
free base
Key result
Sex:
not specified
Dose descriptor:
approximate LD50
Effect level:
ca. 150 mg/kg bw
Based on:
other: caculation
Remarks:
sulfate salt
Mortality:
Genetic toxicity (invivo)_71339_C337906-37-3_E474-270-7_QCE08June11
Range finding: all animals died after single injection of 2000mg/kg bw and 250mg/kg bw doses. All animals survived after a single injection of 100mg/kg bw
Main experiment: no death after injection of a single dose of 100mg/kg bw

Genetic toxicity in vivo (UDS)_60029_C337906-37-3_E474-270-7_QCE08June11
Range finding: at the highest dose (500 mg/kg bw) two out of four animals died, at 200 mg/kg bw one ot of four animals died within 4 hours. No death were observed in the group receiving 100 mg/kg bw.
Main experiment: after a single dose of 75 or 150 mg/kg bw no death were observed

Genetic toxicity in vivo (comet assay)_C337906-37-3_E474-270-7_QCE15June11
Range finding: all animals died shortly after aplication (2000 mg/kg bw oral gavage). Two other groups received a dose of 200 and 100 mg/kg bw respectively. In the first group (200 mg/kg bw) two out of 3 animals died within 19hr, none of the animals died in the second group (100 mg/kg bw)
Main experiment: MBB was administered twice (20h interval) at dosed of 25, 50 and 100 mg/kg bw, no death were observed

For more details see the corresponding studies in the genetic toxicity in vivo section
Other findings:
LC50 calc oral. 2-METHOXY-METHYL-P-PHENYLENEDIAMINE (free base) = 91.5 mg/kg bw

Conclusions: Based on the above, a scientifically sound LD50 between 100 and 200 was derived for the sulfate salt from the MTD values in the rat, which is in line with the OECD criteria. An oral LD50 of 91.5 mg/kg bw
for the free base was calculated using a conversion factor of 0.61.
For the calculations of the acute dermal and inhalation toxicity potential of 2-METHOXY-METHYL-PPHENYLENEDIAMINE sulfate salt and free base, an oral LD50 of 150 mg/kg bw for the sulfate salt was used.

Any other information on results incl. tables

Table 1: Comparison of study requirements from OECD guidelines for acute oral toxicity and for genotoxicity studies

 Test type  Guideline (OECD)  Animal species  Doses (mg/kg bw)  Animal number  Observation period  Determined LD50 (mg/kg bw)
 

Acute oral toxicity

 

423

 Rat  5, 50, 300, 2000 (Guideline recommendation)

 3/dose (Total: 3 to 12)

 1 to 14 days  

Possible values: 5, 25, 50, 200, 300, 500, 2000, 2500, 5000

 Comet Assay in vivo 489   Rat  Determination of MTD (doses tested: 100, 200, 2000)  (required: min. 3) 3 at 200 4 at 100  2 days LD50: < 200 (at 200 2 of 3 animals died, none died at 100) MTD: 100 
 UDS in vivo  486  Rat  Determination of MTD (doses tested: 100, 150, 200, 500 )  (required: min. 3) 4 (2 m, 2 f)/dose  1 day LD50: ≤ 200 (at 200 1 of 4 animals died but at 150 no animals died) MTD: 150 

Applicant's summary and conclusion

Interpretation of results:
Category 3 based on GHS criteria
Conclusions:
Despite the lack of a standardised acute toxicity study (according to OECD guidelines No. 423/425), the available information on this endpoint is considered to be adequate for the purpose of classification/labelling to ensure occupational safety.
From a scientific point of view and with regard to the animal welfare policy the performance of additional in vivo animal studies for the determination of this endpoint is not justifiable.
Executive summary:

The acute oral toxicity of 2-METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE was determined based on the maximum tolerated dose (MTD) after oral application in in vivo genotoxicity studies in rats carried out with the sulfate salt. The determination of the MTD (with respect to the number of animals used and dose selection) was in line with the OECD guideline 423 for the determination of acute oral toxicity. The approach to determine the acute oral toxicity by using MTD data from in vivo genotoxicity studies is accepted by the German (Ref 19) and EU regulatory agencies and is based on the current state-of-the-art science. The calculated oral LD50 values (LD50, calc, oral) for the sulfate salt and free base of 2- METHOXY-METHYL-P-PHENYLENEDIAMINE SULFATE were determined to be 150 mg/kg bw and 91.5 mg/kg bw, respectively. The LD50, calc, oral for the sulfate salt (150 mg/kg bw) was used for the route extrapolation calculations, since the in vivo toxicity and kinetics assays used in the calculations were also carried out with the sulfate salt.

According to the EU criteria, 2-METHOXY-METHYL-PPHENYLENEDIAMINE SULFATE has to be classified as “toxic, if swallowed”.